Patent Application
20060246520
The present invention relates to compositions and methods for determining or modulating the activity of immune and/or target cells in vitro, ex vivo or in vivo. The invention more specifically relates to methods and compositions for determining sensitivity of cells to the activity of γδT cells, as well as to the use of these compositions and methods for patient screening or selection, therapy improvement, compound selection, etc. The invention also provides compositions and kits suitable for carrying out these methods. The methods may be used in any mammalian subject, preferably any human subject that may benefit from γδT cell-mediated therapy, including patients with tumors, immune or infectious diseases.
Peripheral T lymphocytes classically recognize, through their αβ T cell receptors (TCR), foreign peptidic antigens bound to class I or class II major histocompatibility complex (MHC) molecules. Besides these “conventional” T cells, other subsets expressing either αβ or γδ TCR react with a more heterogeneous set of non-peptidic compounds, either in a native form or in association with conserved MHC-related molecules (Beckman et al., 1994; Moody et al., 1997; Spada et al., 2000).
In humans, the vast majority of peripheral blood γδ T cells use a particular combination of variable regions (Vγ9 and Vδ2) to form their TCR. These Vγ9Vδ2 T cells are activated in a TCR-dependent fashion by several small phosporylated (Constant et al., 1994; Tanaka et al., 1994) or aminated (Bukowski et al., 1999) alkyl molecules. Vγ9Vδ2 T cell activation by these compounds requires intercellular contact, thus suggesting some form of antigen presentation (Lang et al., 1995; Morita et al., 1995). Vγ9Vδ2 cells also react against several fresh or cultured tumors in vitro and exhibit both cytolytic activity and production of inflammatory cytokines (TNFα, IFNγ). This activity is tightly regulated by NK-like receptors for MHC class-Ia and class Ib antigens which are prominently expressed by this T cell subset (Fisch et al., 1997; Halary et al., 1997). Thus, besides their role in immunity against viral and bacterial infections (Bukowski et al., 1994), γδ T cells are probably involved in tumor surveillance (Bukowski et al., 1995; Fisch et al., 1997; Wu et al., 2002) as also supported by in vivo experiments (Girardi et al., 2001; Malkovska et al., 1994; Malkovska et al., 1992).
Although some human γδ T cells of the Vδ1 subset react towards non classical, stress-induced MHC molecules MICA/B (Groh et al., 1998; Wu et al., 2002) through their TCR and/or activatory receptors such as NKG2D, tumor antigens recognized by Vγ9Vδ2 T cells remain unknown and strategies for activating these cells, as well as for screening target cells sensitive to γδ T cells' activity are highly needed to develop powerful therapeutic strategies.
The present invention now discloses novel, improved strategies for generating, regulating or exploiting the activity of γδ T cells. The present invention unexpectedly shows that an entity related to the mitochondrial ATP-synthase is expressed on the membrane of target tumor cells and promotes their recognition by Vγ9Vδ2 T cells. When immobilized, purified ATP synthase induces the selective activation of this cell population. The Vγ9Vδ2 TCR and the ATP synthase also bind a delipidated form of apolipoprotein A-I as demonstrated by surface plasmon resonance. Moreover, the presence of apolipoprotein A-I in the culture medium is required for optimal activation of Vγ9Vδ2 T cells and TCR binding to tumors expressing ATP synthase. This invention thus identifies an unanticipated tumor recognition mechanism by Vγ9Vδ2 lymphocytes and allows the design of novel therapeutic and pharmacogenomic approaches.
A first aspect of this invention resides more particularly in methods of assessing the sensitivity of a pathologic cell to γδT cell activity (e.g., lysis or cytotoxicity), comprising determining in vitro or ex vivo whether said pathologic cell expresses, at the surface thereof, an ATP synthase polypeptide or a portion thereof, the cell surface expression of an ATP synthase polypeptide or a portion thereof being an indication of the sensitivity of the pathologic cell to γδT cell lysis or cytotoxicity.
An other aspect of this invention resides in methods of assessing whether a cell is a target cell for γδT cells, comprising determining in vitro or ex vivo whether said cell expresses, at the surface thereof, an ATP synthase polypeptide or a portion thereof, the cell surface expression of an ATP synthase polypeptide or a portion thereof being an indication that the cell is a target of γδT cells.
A further aspect of this invention lies in methods of assessing the responsiveness of a patient to γδT cell-mediated therapy, comprising determining whether said patient contains pathologic cells, tissues or organs that express, at the surface thereof, an ATP synthase polypeptide or a portion thereof, the cell surface expression of an ATP synthase polypeptide or a portion thereof being an indication of the responsiveness of the patient to γδT cell-mediated therapy.
The invention also relates to methods of selecting patients for a γδT cell-mediated therapy program, comprising determining whether a patient contains pathologic cells, tissues or organs that express, at the surface thereof, an ATP synthase polypeptide or a portion thereof, the cell surface expression of an ATP synthase polypeptide or a portion thereof being a criteria for selecting said patient for said γδT cell-mediated therapy program.
A further object of this invention resides in methods of stimulating γδT cells, comprising contacting γδT cells with an ATP synthase polypeptide or a TCR-binding fragment thereof. In a specific embodiment, the stimulation method uses a complex comprising an ATP synthase polypeptide or a TCR-binding fragment thereof linked to an apolipoprotein A1 polypeptide. These components (or some of them) may be immobilized on a solid support, such as a bead, plate, etc.
A further object of this invention resides in methods of stimulating γδT cells, comprising contacting γδT cells with an apolipoprotein A1 polypeptide or a variant or ATP synthase-binding fragment thereof.
The invention also provides a method of increasing sensitivity of a target cell to γδT cell lysis or cytotoxicity, comprising causing or increasing cell surface expression of an ATP synthase polypeptide or a portion thereof in said target cell. The method may be performed in vitro, ex vivo or in vivo.
The invention also relates to an improvement in methods of treating a cancer, an infectious disease, an autoimmune disease or an allergic disease in a subject by γδT cell-mediated therapy, the improvement comprising determining, prior to or during said therapy, whether said subject contains pathologic cells, tissues or organs that express, at the surface thereof, an ATP synthase polypeptide or a portion thereof, the cell surface expression of an ATP synthase polypeptide or a portion thereof being a criteria for selecting or monitoring said patient for said γδT cell-mediated therapy.
The invention also relates to methods of screening, selecting or identifying a compound, comprising:
The invention is particularly suited for use with human cells or subjects, and can be used to diagnose or select cells or patients having cancers or infectious diseases sensitive to γδT cell-mediated therapy. As will be discussed further, the ATP synthase polypeptide may be an ATP synthase α subunit or β subunit or both, and the portion thereof may be an extracellular domain of an ATP synthase α or β subunit.
(a) Indirect immunofluorescence staining with M5 mAb or a control IgG1 antibody (shaded histograms) of human cell lines cultured in presence of fetal calf serum (fcs). Daudi, Raji: Burkitt's lymphomas; Awells, RPMI8866: B-lymphoblastoid cell lines; RPMI 8226: B-cell myeloma; K562: erythroid leukemia; U937: monocytic leukemia; MOLT-4, Jurkat: T cell leukemias.
(b) Daudi cells were depleted of serum components by overnight culture in serum free medium (sfm) and subsequently incubated in PBS supplemented with either 10% fcs (sfm/fcs), 10% human serum (sfm/hs) or 1% BSA plus 100 μg/ml of M5L (sfm/M5L) before staining as in
(c) Serum-depleted cell lines were incubated with lipid-free HDL-apoA-I (solid lines and shaded histograms) or BSA (dotted lines) before indirect staining with the anti-human apoA-I antibody 4H1 (lines) or control IgG1 (shaded histograms).
(d) Left: coomassie-blue staining of M5-immunopurified material from fcs (lane 1) and human serum (lane 2) after non-reducing SDS-polyacrylamide gel electrophoresis (PAGE). Right: Immunoblot after reducing PAGE using anti-human apoA-I antibody 4H1 on: human serum (lane 3); fetal calf serum (lane 4); HDL (lane 5); hM5L human serum ligand of M5 mAb, lane 6).
(e): 125I-labelled free apoA-I binding was measured after 2 hours incubation at 4° C. on Daudi and Raji cells. Top and bottom panels: binding isotherm of 125I-labeled free apoA-I to Daudi (top) and Raji (bottom) cells. (◯): total binding, (σ): non-specific binding; (●): specific binding. Middle panel: Scatchard representation of specific binding on Daudi cells. In absence of specific binding on Raji cells, Scatchard representation could only be performed from Daudi cells data The results are representative of two different cell preparations.
(a,b) Indirect immunofluorescence surface staining of haematopoietic tumor lines with (a) anti α-AS and (b) anti-β-AS. (c) Four kidney tumours sensitive to γ9δ2 lysis were tested for expression of AS by facs staining using control IgG (shaded histogram), anti-α-AS (dark line) and anti-β-AS (dotted line).
(a) T cell clones were activated with protein-coated beads in medium supplemented with human serum, and TNFα secretion was measured in the culture supernatant after 4 hours. The capacity of γδ clones G25, G115, 73R9 to secrete significant amounts of TNFα upon stimulation was checked by using Daudi cells as target cells as well as PMA/ionomycin or PHA. The αβ clone A4.19 secretes saturating quantities of TNFα upon PMA/ionomycin or PHA stimulation (not shown).
(b) Stimulation was performed in the absence of exogenous serum (sfm) and purified HDL-derived apoA-I was added in some cultures (black bars).
(c) Fresh PBL were activated with indicated beads for 4 hours in the presence brefeldin A. Cells were then stained with antibodies to lymphocyte subsets, fixed, permeabilized and TNFα accumulation was analyzed after intracellular staining by flow cytometry. PMA/ionomycin stimulation is used as a control for TNFα-producing cells in the total population. Percentages indicating TNFα-producing cells within analysis quadrants and within each particular subset (between brackets) are shown.
(a,b) Overlay sensorgrams for SPR analysis of soluble TCRγδ protein binding to immobilized apoA-I. Amount of immobilized ApoA-I protein was 2260 RU on flow cell 2 (a): comparative sensorgrams of soluble Vγ9Vδ2TCR and Vγ8Vδ3TCR (200 and 2000 nM) binding onto immobilized apoA-I. (b) Vγ9Vδ2TCR was injected at concentrations ranging from 125 nM to 2 μM. The apparent kinetic constants of the interaction were ka=8.8e3±6.08 e2 M−1, kd=7.13 e−3±6.76 e−5s−1, KD=8.1 e−7M.
(c) Comparative sensorgrams of purified apoA-I (full line) and apoA-II (dotted line) binding to immobilized Vγ9Vδ2TCR (proteins were injected at 100 μg/ml). Amount of immobilized TCRγδ protein was 335 RU on flow cell 2.
(d,e) Comparative sensorgrams of soluble TCR binding to immobilized ATP synthase. Amounts of immobilized ATP synthase (F1) was 22440 RU on flow cell 2. (d) Comparative sensorgrams of purified monomeric G115 TCR (Vγ9Vδ2, full line) and 73R9 TCR (Vγ8Vδ3, dotted line) binding to immobilized ATP synthase (F1). Proteins were injected at a concentration of 2 μM. (e) Monomeric G115 soluble TCR (Vγ9Vδ2) was injected at concentrations ranging from 0.5 to 4 μM. The apparent kinetic constants of the interaction were ka=1.68 e3±2.16 M−1s−1, kd=2.54 e−3±2.9 e−5s−1, KD=1.51 e−6M.
T cells of the γδ type are expressed by most mammalian species. They represent 1-10% of total circulating lymphocytes in healthy adult human subjects and most non-human primates. Most human peripheral blood γδ T cells express a γδTCR heterodimer encoded by Vγ9/Vδ2 genes, some NK-lineage receptors for MHC class I and almost no CD4 nor CD8. These cells have been shown to exhibit strong, non MHC-restricted, cytolytic activity against virus-infected cells (Poccia et al, J. Leukocyte Biology, 62, 1997, p. 1-5), parasite-infected cells (Constant et al, Infection and Immunity, vol. 63, n° 12, December 1995, p. 4628-4633), or tumor cells (Fournie et Bonneville, Res. Immunol., 66th FORUM IN IMMUNOLOGY, 147, p. 338-347). These cells are also physiologically amplified in the context of several unrelated infectious diseases such as tuberculosis, malaria, tularemia, colibacillosis and also by B-cell tumors (for review see Hayday, 2000). These cells are thus viewed as potent effectors of innate immunity and represent an important resource of anti-infectious and anti-tumoral effectors. In this regard, γδT cell-mediated therapeutic programs are currently under development, particularly for treating cancers, using ex vivo activated cells or direct in vivo injection of γδT activators. However, in order to increase the efficacy of these treatments, it would be highly valuable to have methods available for selecting best responding patients or for selecting target cells, as well as for increasing the sensitivity of target or pathologic cells to these γδT cell-mediated therapies.
The instant invention now provides such methods.
As disclosed above, the invention relates to methods of assessing the sensitivity of cells to the activity of γδT cells, e.g., to their lysis or cytotoxicity. The tested cell may be any cell, preferably a mammalian cell, typically a human cell. The tested cell is generally a pathologic cell (i.e., a cell associated with or resulting from a disease in a subject), and the method is conducted in order to evaluate the efficacy of a γδT cell-mediated therapy against said pathology.
Typical examples of such target or test cells include pathogenic mammalian (e.g., human) tumor cells, pathogen-infected cells, pathologic immune cells, etc. Specific examples of tumor cells include any solid or hematopoietic tumor cell, such as renal cancer cells, liver cancer cells, lung cancer cells, bladder cancer cells, breast cancer cells, colon cancer cells, CNS cancer cells, head-and-neck cancer cells, etc. The sample used may be any isolated pathologic cell, or a tissue, organ, tissue sample, body fluid, etc. In a specific embodiment, the method is carried out on a biopsy from a subject, typically a biopsy section. The method may be performed on a sample collected from a subject and stored, optionally in frozen state, or extemporaneously, upon collection from the subject.
Determination of responsiveness or sensitivity to γδT cell comprises, within the context of the invention, an assessment of cell surface expression of an ATP synthase polypeptide or a fragment thereof. Indeed, the present invention now shows that ATP synthase participates in γδT cell activation, and is directly involved in TCR recognition and binding. The invention also shows that cells that are sensitive to γδT cells express at their surface an ATP synthase, while such protein is normally expressed intracellularly (i.e., essentially in mitochondria). By detecting ATP synthase cell surface expression, sensitivity of a target cell to γδT cells may thus be evaluated.
Within the context of this invention, an ATP synthase polypeptide designates any polypeptide, protein or peptide fragment of an ATP synthase, as well as variants, isoforms and orthologs thereof. The sequence of ATP synthase genes and proteins has been disclosed in the prior art literature, and is available in various gene libraries. In particular, the amino acid sequence of a human ATP synthase protein is available on SwissProt Data base under the following accession numbers: F1-α subunit: P25705; F1-β subunit: PO6576; F1-γ subunit: P36542; F1-δ subunit: 30049; F1-ε subunit: P56381. The nucleotide sequence of a human ATP synthase cDNA is available on GenBank Data base under the following accession numbers: F1-α subunit: BT007209; F1-β subunit: NM01686; F1-γ subunit: XM292254; F1-δ subunit: NM001687; F1-ε subunit: BT007293. Further information regarding the cloning, sequence, characteristics, expression and structure-function of a human ATP synthase gene or protein can be found in Kataoka, H. and Biswas, C., Biochim. Biophys. Acta 1089 (3), 393-395 (1991); Neckelmann, N., et al., Genomics 5 (4), 829-843 (1989); Matsuda, C., et al., J. Biol. Chem. 268 (33), 24950-24958 (1993) and Biochim. Biophys. Acta 1130 (1), 123-126 (1992) or Tu, Q., et al., Biochem. J. 347 Pt 1, 17-21 (2000), incorporated therein by reference.
The mitochondrial ATP synthase is typically composed of an intramembrane sector termed F0 and an extramembrane sector termed F1. The F1 sector comprises α, β, γ, δ and ε subunits in an α3β3γδε polypeptidic complex. At least the α and β polypeptides show ectopic expression and can be found at the surface of pathologic cells, such as tumor cells.
Accordingly, in a specific embodiment, the ATP synthase polypeptide is an ATP synthase α subunit.
In an other specific embodiment, the ATP synthase polypeptide is an ATP synthase β subunit
In a further specific embodiment, the ATP synthase polypeptide is a complex formed between an ATP synthase α and β subunits.
In an other specific embodiment, the method comprises detecting a portion of an ATP synthase, typically an extracellular domain of an ATP synthase α or β subunit.
Determination of said cell surface expression can be performed using a variety of techniques known per se, such as by ligand binding, immunohistochemistry, etc. In a specific embodiment, the method comprises incubating the biological sample comprising said test, target or pathogenic cells (e.g., cultured cells, tissue or organ) with a ligand specific for an ATP synthase polypeptide or a portion thereof, and assessing binding of said ligand to said cell, tissue or organ. In a more preferred embodiment, the ligand is an antibody or a fragment or derivative thereof (e.g., a Fab or Fab′2 fragment, a CDR region, a SCfV, etc.). The ligand or antibody may be labelled, e.g., by fluorescent, enzymatic, luminescent, radioisotope, etc. labels, to facilitate binding determination.
The ATP synthase polypeptide, particularly the extracellular domains thereof, may be detected by indirect immnunofluorescence using commercially available antibodies. As a specific example, antibodies specific for the α or β subunits may be found at Molecular Probes, Inc., under the following references:
In a specific embodiment, the method comprises providing a biopsy from the patient and determining cell surface expression of an ATP synthase polypeptide or a portion thereof by immunohistochemistry using an antibody.
As mentioned, the method allows the design of improved or customized therapies, by determining which patients would best respond to particular γδT cell-mediated therapies. In this regard, the γδT cell-mediated therapy may be a direct in vivo therapy (e.g., comprising the injection of a drug that stimulates γδT cells in said patient) or an ex vivo cell therapy (e.g., comprising the injection of ex vivo activated γδT cells to said patient).
The invention also provides novel methods of stimulating γδT cells, comprising contacting γδT cells with an ATP synthase polypeptide or a TCR-binding fragment thereof. Alternatively, the invention also provides methods of stimulating γδT cells comprising contacting γδT cells with an apolipoprotein A1 polypeptide or a variant or ATP synthase—or TCR-binding fragment thereof. The present invention indeed discloses that Apolipoprotein A-I is a ligand of ATP synthase and of the TCR of lymphocytes Tγ9δ2, so that this polypeptide or, more preferably, derivatives thereof, represent valuable therapeutic agents to mediate γδT cell therapy. These derivatives are preferably designed to have an increased affinity for the cell surface-expressed ATP synthase and/or for the TCRγ9δ2, allowing the specific targeting of this lymphocyte population toward pathologic (e.g., tumor) cells.
These stimulation methods can be carried out in vivo, in vitro or ex vivo.
In a specific embodiment, the method comprises contacting γδT cells with a complex comprising an ATP synthase polypeptide or a TCR-binding fragment thereof linked to an apolipoprotein A1 polypeptide. In a further preferred embodiment, the ATP synthase polypeptide or fragment is immobilized on a support. Indeed, as shown in the examples, solid support coated with ATP synthase polypeptide is able to stimulate γδT cells. The support may be a bead, column, plate, etc. The γδT cells are preferably human cells.
In this respect an object of this invention resides in a product comprising, immobilized on a support, an ATP synthase polypeptide or a TCR-binding fragment thereof.
A further object of this invention resides in a method of increasing sensitivity of a target cell to γδT cell lysis or cytotoxicity, comprising causing or increasing cell surface expression of an ATP synthase polypeptide or a portion thereof in said target cell.
A further object of this invention is a method of screening, selecting or identifying a compound, comprising:
The method is typically conducted in vitro, in any suitable device, such as a plate, tube, flask, pouch, etc. The method is particularly suited for use in multi-well plates, to screen several compounds in parallel. In the above screening methods, the ATP synthase polypeptide or TCR-binding fragment thereof may be in suspension or immobilized on a support, such as a bead or at the surface of the device itself. Also, the method may be conducted in the presence of an apolipoprotein A1 polypeptide. Furthermore, the method is suitable for selecting or designing or improving apolipoprotein A1 polypeptide variants that modulate activation of γδT cells. The activity of γδT cells may be assessed using a number of biological assays, such as proliferation, target cell lysis, cytokine release, etc. The method is particularly suited to identify compounds that activate γδT cells.
Further aspects and advantages of this invention will be disclosed in the following experimental section, which should be regarded as illustrative and not limiting the scope of this application.
1) Tumor Cell Lines, T Cell Clones and Cultures
Daudi, Raji, RPMI 8226, K562, Jurkat, Molt-4, and U937, SK-NEP, G401, G402, and 786.0 were obtained from ATCC. Awells (EBV+ lymphoblastoid B cell line) is from the International Histocompatibility Workshop (IHW#9090). C1R (HLA-A−B−-LCL) and RPMI 8866 (B-LCL) were obtained from Drs P. Lebouteiller and M. Colonna respectively. All tumor cell lines were cultivated in RPMI 1640 medium supplemented with 10% fetal calf serum (Invitrogen) except for serum deprivation experiments. In this case, cells were washed once in RPMI 1640, incubated for 1 hour at 37° C. in serum free culture medium (hybridoma sfm, Invitrogen), pelletted and reincubated for at least 16 hours at 37° C. in sfm before use. The G25 (Vγ9Vδ2) and 73R9 (Vγ8Vδ3) clones were obtained as described for G42 and G115 (Allison et al., 2001; Davodeau et al., 1993) by anti-Vδ monoclonal antibody selection and subsequent amplification using PHA and IL2 and cloning.
3) Fluorescence Analysis and Antibodies
Immunofluorescence stainings were performed in PBS containing 1% BSA and devoid of serum using FITC-conjugated goat F(ab)′2 anti-mouse Ig antibody (Caltag) as the second step reagent. Irrelevant isotype-matched control antibodies were used as negative controls. In apolipoprotein binding experiments, serum-deprived cells were incubated with serum or purified protein preparations in PBS containing 1% BSA, at room temperature, 30 minutes prior to antibody staining. 7H10 (anti-α-ATP synthase) and 3D5 (anti-β-ATP synthase) and 7F9 (anti-γ-ATP synthase) are from Molecular Probes. 4H1 (anti-human apoA-I) (Collet et al., 1997) was obtained from Dr Y. Marcel (Ottawa).
4) Generation of M5A12D10 Hybridoma and Antibody Purification
Balb/c mice were injected intraperitoneally four times at two-weeks intervals with 15×106 Daudi cells washed and resuspended in PBS. Hybridoma were obtained by fusion of spleen cells with P3X63Ag8 myeloma cells and were selected on the basis of tumor cell staining. Subcloned hybridoma were subsequently amplified in sfm medium and antibodies were purified on protein G affinity columns (Pharmacia), neutralized, dialyzed against PBS and concentrated (Harlow and Lane, 1988). Finally, antibodies were tested for their ability to modulate lysis of Daudi cells by γδ effectors, leading to the selection of the M5A12D10 antibody (IgG1).
5) Purified Proteins
The human and bovine ligands of M5A12D10 (hM5L and hM5L respectively) were isolated by affinity chromatography: human and fetal calf serum diluted 1/20 in 3 M NaCl and 50 mM Tris pH 7 were passed through the column carrying the covalently attached antibody. After washing (last wash was in 3 M NaCl, 10 mM Tris pH 7), bound proteins were eluted with 100 mM glycine pH 2.7. Isolation of ApoA-I from High Density Lipoproteins (HDL apoA-I) by ion-exchange chromatography has been already described (Mezdour et al., 1987). Purity of apoA-I was checked by Western blot analyses using different antibodies directed against human apoB, apoA-II, apo-C and apoA-I. The apolipoprotein A-I homogeneity was more than 99% (as measured by densitometry after SDS-PAGE and silver staining). Purified bovine ATP synthase (F1 subunit) (Lutter et al., 1993) was obtained from John E. Walker (Cambridge, UK).
6) Production of Soluble Fluorescent Tetrameric TCR and Usage for Cell Staining
The G115 (Vγ9Vδ2) (3) and 73R9 (Vγ8Vδ3) extracellular γ and δ chains (the latter carrying a short 3′ Biotin tag) were expressed in Escherichia coli, then refolded together by rapid dilution in 1 L of 1 M L-arginine, 0.1 M Tris-HCl, pH 8.0 and 0.2 mM reduced/0.2 mM oxidized glutathione. After dialysis against 10 mM Tris-HCl pH 8.0, concentration by cation exchange chromatography at pH 5.5, and purification by size exclusion chromatography at pH 8.0, the refolded protein was biotinylated for 4 h at 30° C. with 6 μg/ml BirA and excess of free biotin was removed by dialysis against 10 mM Tris-HCl pH 8.0, 150 mM NaCl. Tetramers of γδ TCR heterodimers were obtained by mixing the biotinylated TCR with phycoerythrin-labeled streptavidin (Biosource) at a molar ratio of 10:1. For staining, 2.105 cells were incubated at room temperature for 45 min with phycoerythrin-labeled γδ TCR tetramers at a concentration of TCR of 30 μg/ml in PBS plus 1% BSA. Background staining was determined using phycoerytlrin-labeled streptavidin at the same concentration.
7) Protein Identification by Proteomic Analysis and Mass Spectrometry
Immunopurified material was analyzed using one dimensional electrophoresis and visualized by coomassie blue staining. Protein bands were excised from the gel and subjected to several washing steps, reduction alkylation reaction, in-gel trypsin digestion with modified trypsine (Promega, Madison, Wis.) at 25 ng/μl in 50 mM NH4HCO3 and finally followed by peptide extraction. The peptides purified with ZipTip C18 (Millipore) were mixed with equal volumes (0.5 ml) of a saturated a-cyano-4-hydroxycinnamic acid in 50% acetonitrile, 0.1% TFA onto the MALDI target and allowed to air-dry. Peptide mass fingerprinting were obtained by using a PE Biosystems MALDI-TOF mass spectrometer (Voyager DE STR, Foster City, Calif., USA) on each protein band. Unknown proteins were identified using the data base fitting program MS-Fit (Protein Prospector, (http://prospector.ucsf.edu)), searching against all eukaryotic entries in Swiss Prot and NCBI non redundant protein data bases. We considered the identifcation positive when a minimum of four measured peptide masses were matched and provided at least around 20% sequence coverage. Mass accuracy of 10 ppm was obtained with internal calibration using auto-digestion peaks of trypsin (M+H+, 842.51, 2211.10, and 2283.18).
8) Chromium and Cytokine Release Assays
2 h- 51Cr-release assays were performed in standard conditions except for the use of serum-free conditions in some experiments: in sensitization experiments with apolipoprotein preparations, target tumor cells were serum-deprived as described for facs analysis, loaded with 51Cr (100 μCi/106 cells, 1 hour, 37° C.), extensively washed in RPMI and resuspended in sfm medium. Effector T cells cultivated in serum-containing medium were washed extensively in RPMI, incubated for 2 hours in sfm at 37° C. and resuspended in sfm medium. Target cells (3000/well, in triplicates) were first incubated with apolipoprotein preparations or serum-containing medium for 30 min at room temperature in 96-well round-bottom microculture plates at room temperature prior to the addition of effector cells. Cells were then pelleted and incubated at 37° C. for 2 hours. Supernatants were recovered for 51Cr release measurement. Spontaneous release (in the absence of effectors) was subtracted from experimental data and was in the 10-30% range of maximum release (effector cells replaced by same volume of 0.1 M HCl). Specific lysis was calculated as the percentage of maximum release. For cytokine release measurements, similar experiments were performed supernatants were harvested after 4-hours (TNFα) or 20 hours (IFNγ). IFNγ was titrated by a specific Elisa technique, whereas TNFα concentration was assessed by a biological assay based on WEHI cells viability.
9) Binding Assays
The binding experiments were performed at 4° C. for 2 hours as previously described (Barbaras et al., 1994). Briefly, cells (9 μg of cell proteins per point) were incubated in PBS for 2 hours at 4° C. with increasing concentrations of labeled apoA-I. Cells were filtered on 0.22 μm filters (GVWP Millipore-France) and washed four times with 1% BSA in PBS. Filters were used for radioactivity measurements. Non-specific binding was determined in the presence of a 100-fold excess (as compared to the KD value) of the corresponding unlabeled ligand. Binding was analyzed using a weighted non-linear curve-fitting program, based on the LIGAND analysis program (Prism-GraphPad).
10) Immobilization of Proteins on Latex Beads.
107 sulfate latex beads (Interfacial Dynamics corp., Portland, Oreg.) were washed in PBS and incubated with apolipoproteins (100 μg/ml), F1-ATP synthase (0.4 mg/ml) or a mixture of both under constant agitation at room temperature for 16 hours. Beads were then washed, saturated for 3 hours in PBS containing 1% BSA and washed extensively in PBS before use. Control “empty” beads were similarly saturated with BSA. In stimulation experiments, beads were mixed with cells at a 1:1 ratio.
11) Immobilization of Proteins for SPR (Biacore) Analysis.
ApoA-I and ATP synthase (F1 subunit) were immobilized by amine linkage on CM5 chips (Biacore AB) following NHS-EDC activation; Vγ9Vδ2-biotinylated soluble TCRs were immobilized on SA streptavidin-coated chips (Biacore AB) and binding was analyzed in a Biacore 3000 apparatus (BiacoreAB). Soluble ligands were injected at a flow rate of 20 μl/min, exposed to the surface for 240 s (association phase) followed by a 240-s flow running during which the dissociation occurred. Sensorgrams are representative of specific interactions (differential response) where non-specific binding that occurred on flow cell 1 was deduced from binding that occurred on flow cell 2. Results are expressed as resonance units (RU) as a function of time in seconds.
Results
In an attempt to characterize such antigens, we raised murine monoclonal antibodies against the Burkitt's lymphoma Daudi, a cell line which is lysed by a large majority of Vγ9Vδ2 T cell clones (Davodeau et al., 1993; De Libero et al., 1991). We selected monoclonal antibodies (mAb) for their differential binding to Daudi and Raji (a non-activating Burkitt's lymphoma) and for their ability to interfere with Vγ9Vδ2 T cell recognition of Daudi cells. One mAb (#M5A12D10, hereafter referred to as M5) which fulfilled both criteria was selected for further studies. M5 mAb stained Daudi cells as well as several other hematopoietic tumors reported to be recognized by Vγ9Vδ2 T cells, including cell lines of lymphoid (MOLT-4, RPMI 8226) and myeloid (K562, U937) origins. By contrast several tumor cells resistant to Vγ9Vδ2 T cell killing such as Raji and B lymphoblastoid cells were not stained by M5 mAb (
Modulation of Vγ9Vδ2 T cell activity by apo A-I could be due to apoA-I recognition by either the Vγ9Vδ2 TCR itself or by accessory receptors such as toll-like receptors which are known to be expressed on various conventional and non-conventional T cell subsets (Caramalho et al., 2003; Mokuno et al., 2000; Sakaguchi, 2003). Possible involvement of apoA-I in TCR engagement was studied by using recombinant soluble forms of the Vγ9Vδ2 TCR derived from the G115 clone (Allison et al., 2001), which were biotinylated and tetramerized by fluorescent streptavidin (hereafter referred to as TCR tetramers). Like M5 mAb, Vγ9Vδ2 TCR tetramers bound to the Vγ9Vδ2 target cells Daudi, K562 and more weakly to RPMI 8226, but neither to Raji cells nor to B lymphoblastoid cell lines (
We have recently shown that components of the mitochondrial enzyme ATP-synthase (AS) can be expressed on the surface of hepatocytes, and that this enzyme constitutes a high affinity receptor for free, delipidated apoA-I on these cells (Martinez et al., 2003). We studied expression of components of this enzyme on the surface of Vγ9Vδ2 tumor targets. Daudi, K562 and RPMI 8226 were stained by mAb against the a chain of AS (αAS) whereas Raji, leukaemic T cells and B-LCL were not (
Two sets of experiments were performed to assess the involvement of AS in tumor cell recognition by Vγ9Vδ2 T cells. Firstly, antibodies to apoA-I and to the extramembrane (F1) subunit of AS were tested for their ability to modulate lymphokine secretion by Vγ9Vδ2 T cells following incubation with Daudi cells. IFNγ secretion was strongly inhibited by anti-αAS, anti-βAS and M5, as compared to a control Ig. This inhibition was not merely due to Ab toxicity since Vγ9Vδ2 T activation was largely restored by addition of isopentenyl pyrophosphate (IPP), a previously described Vγ9Vδ2 T cell antigen (
Secondly, the involvement of AS in Vγ9Vδ2 T cell activation was studied by testing the ability of mitochondrial bovine AS immobilized on latex beads with and without apoA-I to stimulate Vγ9Vδ2 T cells. Beads carrying apoA-I only or none of these proteins were also tested (
Altogether the above results suggested a highly specific and TCR-mediated activation of Vγ9Vδ2 cells by apoA-I and AS. However, the respective contribution of AS and apoA-I in γδ T cell/tumor cell interactions remained unclear. Although anti-apoA-I antibodies had a substantial inhibitory effect on cytotoxicity and lymphokine secretion, these effects as well as the modulatory properties of apoA-I could be explained by the proximity of apoA-I and AS on the cell surface, possibly generating indirect perturbation of TCR-AS interactions. Surface plasmon resonance analysis (SPR) has been previously used to demonstrate interactions between apoA-I and ATP synthase (Martinez et al., 2003). We followed a similar approach to assess TCR/apoA-I/AS interactions, after immobilization of either apoA-I, soluble G115 TCR or purified AS onto SPR chips. Immobilized apoA-I specifically interacted with soluble monomeric G115 TCR (KD{tilde over ( )}0.8 μM) whereas interaction with the 73R9 TCR was too low to be measured (
Discussion
Our results strongly suggest that during interaction between Vγ9Vδ2 T cells and their tumoral targets, a ternary complex forms between the TCR on the T cell side and apoA-I plus AS on the tumor cell side. It is not clear whether the structure expressed on the surface of tumors is identical to mitochondrial AS: reactivity with available monoclonal antibodies suggests that the αAS and βAS chains can be independently expressed on various tumors, indicating some form of surface expression polymorphism of these proteins. Using a limited panel of tumor lines we found that sensitivity to Vγ9Vδ2 T cells correlated better with αAS than with βAS expression. Although AS might not be the primary tumor target antigen of Vγ9Vδ2 T cells but instead a cross-reactive unrelated entity, the stimulatory potential of immobilized mitochondrial AS would indicate that its homology to the putative primary antigen(s) is high enough to induce a potent Vγ9Vδ2 T cell stimulation.
Direct binding of apoA-I to AS (Martinez et al., 2003) and the TCR (our present data) have now been demonstrated. These interactions seem to be required for optimal T cell activation as Vγ9Vδ2 T cell activation and soluble TCR binding were markedly decreased by serum depletion and were restored by addition of exogenous soluble apoA-I. However, our present results suggest a hierarchical importance of these interactions for Vγ9Vδ2 T cell activation. Indeed, we could not demonstrate any stimulatory activity for immobilized apoA-I whereas AS appeared to be stimulatory by itself when immobilized on latex beads. SPR experiments however strongly suggest a direct interaction of apoA-I with both the TCR and AS. Thus a possible role for apoA-I could be the stabilisation of the interaction between the TCR and the AS, explaining the stimulatory effect of apoA-I in cytotoxicity assays.
ApoA-I is unlikely to be immunogenic by itself and, as already mentionned, immobilized apoA-I did not activate Vγ9Vδ2 T cells. However, apoA-I may serve as a carrier for antigenic ligands such as phosphoantigens, either derived from the tumor cells themselves or from the extracellular medium, thus permitting their “presentation” by a surface AS-related structure. This possibility would be consistent with the recently described correlation between endogenous production of mevalonate pathway metabolites (including IPP) by tumor cells and their susceptibility to Vγ9Vδ2 T cell-mediated lysis (Gober et al., 2003).
Ectopic expression of components of AS is not unprecedented as the presence of AS was previously described on the surface of K562 cells (Das et al., 1994) and endothelial cells (Moser et al., 1999) and linked to immunomodulatory effects in one case. The α/βAS subunits are also found on hepatocytes where they promote binding of free apoA-I and display enzymatic activity (Martinez et al., 2003). Whether these components are also involved in an enzymatic complex on tumor cells is not known yet. Although expression of this apoA-I receptor on normal tissue cells could potentially induce Vγ9Vδ2 T lymphocyte-mediated autoimmunity, several recent studies have described mechanisms involving NK-like receptors and permitting efficient control of this potential self reactivity (Fisch et al., 1997; Halary et al., 1997).
Besides, together with the high expression of LDL receptors and apoE in intraepithelial γδ lymphocytes (Fahrer et al., 2001), and with studies documenting apoE binding to ATP-synthase (Mahley et al., 1989), these data open a new field of investigations linking lipid metabolism and anti-tumor immunosurveillance.
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| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP04/05408 | 5/19/2004 | WO | 11/22/2005 |
| Number | Date | Country | |
|---|---|---|---|
| 60472735 | May 2003 | US |
