Human mast cell lines, preparation and uses

Abstract

The present invention relates to a human mast cell line corresponding to deposit number CNCM I-4551 and also to the lines derived therefrom, in particular the derived lines corresponding respectively to deposit numbers CNCM I-4552 and CNCM I-4553, and to the uses thereof, in particular for screening for compounds of therapeutic interest.

Claims

1. A human mast cell line having the following morphological, ultrastructural and phenotypic characteristics: a) presence of metachromatic intra-cytoplasmic granulations, and b) expression of FcR1 and wild-type KIT (CD117) receptors, and of CD33, CD203c and CD300a markers, and also the following functional characteristics, strict dependence with respect to SCF for its survival and its growth; a doubling time of at most 72 hours; and proliferation for a period of at least six months, wherein said human mast cell line is identified as ROSA KIT WT as registered under deposit number CNCM I-4551 with the Collection Nationale de Cultures de Micro-organismes (CNCM) on 2 Nov. 2011.

2. A cell line derived from the human mast cell line identified as ROSA KIT WT according to claim 1, said derived cell line being identified as ROSA KIT D816V as registered under deposit number CNCM I-4552 with the CNCM on 2 Nov. 2011.

3. A cell line derived from the human mast cell line identified as ROSA KIT WT according to claim 1, said derived cell line being identified as ROSA KIT Delta 417-419 insY as registered under deposit number CNCM 1-4553 with the CNCM on 2 Nov. 2011.

4. A kit for screening for an agent of interest, comprising i) the mast cell line according to claim 1 and; ii) a supplementary product chosen from one or more culture media, one or more maintenance media, one or more growth factors enabling or promoting the culturing of the mast cells; and any combination of said products.

5. A kit for screening for an agent of interest, comprising i) the mast cell line according to claim 2 and; ii) a supplementary product chosen from one or more culture media, one or more maintenance media, one or more growth factors enabling or promoting the culturing of the mast cells; and any combination of said products.

Description

FIGURE LEGENDS

(1) FIG. 1: Characterization of the ROSA KIT WT cells by May-Grnwald Giemsa (MGG) staining, toluidine blue staining and Transmission Electron Microscopy (TEM).

(2) FIG. 2: Expression of the KIT receptor (left-hand histogram) and of the high-affinity IgE receptor (FcR1) by the cells of the ROSA KIT WT line (flow cytometry analysis).

(3) FIG. 3: Demonstration of the tryptase content and HDC content of the cells of the ROSA KIT WT line by the immunocytochemical method on cytospun cells.

(4) FIG. 4: Flow cytometry study of various compounds, including IL-4, on the expression of KIT and of FcR1 by the cells of the ROSA KIT WT line.

(5) FIG. 5: Flow cytometry study of the effect of various treatments on the expression of membrane CD203c by the cells of the ROSA KIT WT line.

(6) FIG. 6: Measurement by the spectrophotometric method of the -hexosaminidase activity released by the cells of the ROSA KIT WT line after stimulation thereof.

(7) FIG. 7: Measurement of the percentage of histamine released by the cells of the ROSA KIT WT line after stimulation thereof by the IgE-anti-IgE pairing.

(8) FIG. 8: Measurement of the TNF- released into the supernatant of the cells of the ROSA KIT WT line after stimulation thereof for 6 hours with the IgE-anti-IgE pairing.

(9) FIG. 9: Morphological and phenotypic appearance of the cells of the ROSA KIT WT line cocultured for more than three months on a sublayer of MS-5 mouse lipoblast cells (A: MGG staining; B: toluidine blue staining; C: immunocytochemical labelling of chymase; D: flow cytometry analysis of various membrane markers).

(10) FIG. 10: Measurement of the infection efficiency by flow cytometry (% of cells positive for GFP) four days after contact of the ROSA KIT WT cells with the various lentiviral constructs used at increasing concentrations.

(11) FIG. 11: Morphological appearance of the cells of the ROSA KIT Delta 417-419 insY and KIT D816V lines after MGG staining. Presence of immature cells, without any granulation for the ROSA KIT Delta 417-419 insY line (red arrow), while in the ROSA KIT D816V line, cells undergoing apoptosis are observed, with numerous granules.

(12) FIG. 12: Flow cytometry study of the expression of certain membrane markers by cells of the ROSA KIT Delta 417-419 insY and KIT D816V lines treated or not treated with SCF (80 ng/ml) and by cells of the ROSA KIT WT line.

(13) FIG. 13: Example of the appearance of a culture in semi-solid medium (1% methylcellulose) of the cells of the ROSA KIT Delta 417-419 insY line after incubation for 15 days in a Petri dish. The colonies, of variable sizes, are identifiable to the naked eye.

(14) FIG. 14: Demonstration by Western blotting of the spontaneous phosphorylation of KIT in the HMC-1.2, ROSA KIT D816V and ROSA KIT Delta 417-419 insY lines. The cell lysates prepared from the nonstimulated cells or the cells stimulated for 5 minutes with human SCF were subjected to SDS-PAGE and treated with an anti-P KIT Y703 antibody (Cell signalling, ref: 3073S). The membranes were subsequently dehybridized and then rehybridized with an anti-KIT antibody (Santa Cruz Biotechnology, ref: J2709).

(15) FIG. 15: Demonstration of the effect of tyrosine kinase inhibitors on the proliferation of the ROSA KIT WT, ROSA KIT Delta 417-419 insY and ROSA KIT D816V lines. The values presented are compared with those obtained without inhibitor. The results represent the mean +/ standard deviation of triplicates.

(16) FIG. 16: Structure of the KIT receptor (CD117) and principal abnormalities encountered during mastocytoses.

(17) FIG. 17: Effect of the specific inhibition of AKT on the proliferation of the two ROSA cell lines.

(18) The ROSA.sup.KIT WT () or ROSA.sup.KIT D816V ( - - - ) cells are seeded for 48 hours in the presence of variable concentrations (0.01 to 5 M) of the powerful and selective inhibitor of AKT1/AKT2, the A6730 compound (diluted in DMSO to a final concentration of 0.1%), or in the presence of DMSO alone (final concentration of 0.1%) in SFM (with rhSCF at 80 ng/ml for the ROSA KIT WT cells but not for the ROSA KIT D816V cells). At the end of this incubation period, 10 l of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were added to each well and the cells were incubated for a further 3 hours in an incubator at 37 C. The number of live cells was then measured for each condition by reading the absorbance at 450 nm. The data given in FIG. 17 represent the meanthe standard deviation obtained from 3 independent experiments and are expressed as percentage proliferation for each condition relative to the control (DMSO alone) representing 100% proliferation.

(19) FIG. 18: Effect of three tyrosine kinase inhibitors on the proliferation of the two ROSA cell lines.

(20) The cells were seeded for 48 hours in the presence of imatinib, dasatinib or midostaurin (1 M, supplied in DMSO at a final concentration of 0.1%) or in the presence of DMSO alone (final concentration 0.1%) in a conventional culture medium (containing human SCF at 80 ng/ml for the ROSA.sup.KIT WT cells but without SCF for the ROSA.sup.KIT D816V cells). At the end of this incubation period, 10 l of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were added to each well and the cells were incubated for an additional 3 hours in an incubator at 37 C. The number of live cells was then measured for each condition by reading the absorbance at 450 nm. The data given in FIG. 18 represent the meanthe standard deviation obtained from 3 independent experiments. ***: value significantly different from the control (DMSO alone) at p<0.0001.

(21) FIG. 19: Demonstration by Western blotting of the spontaneous phosphylation of KIT, AKT and STAT-5 in the ROSA.sup.KIT D816V cells.

(22) The cell lysates originating from nonstimulated ROSA cell lines and from the HMC-1.2 cell line or from identical cells stimulated for 10 minutes with human SCF were subjected to SDS-PAGE and treated with an anti-total human KIT or with an anti-P-KIT Y703 (A), with an anti-total human AKT or an anti-P-AKT S473 (B), or with an anti-total human STAT-5 or an anti-P-STAT-5 Y694 (C). D: an anti-human GAPDH was used as a loading control.

EXAMPLES

Example 1: Isolation, Characterization and Maintenance of Pure Populations of Mast Cells from Normal Human Umbilical Cord Blood: the Rosa KIT WT Line

(23) 1) Sampling and Culturing of Cells

(24) The normal umbilical cord blood sample was collected on lithium heparinate on 9 Sep. 2009. The blood (40 ml) was immediately diluted 50/50 in PBS buffer (Invitrogen) and the mononuclear cells were separated from the other blood components by centrifugation at 700 g for 30 min on a Ficoll gradient (Eurobio). The mononuclear cells, which form a ring at the interface between the Ficoll and the diluted serum, were recovered and washed with PBS buffer. Counting and cell viability were performed using trypan blue (Sigma). The umbilical cord blood haematopoietic progenitors expressing the CD34 surface antigen were then positively selected by immunomagnetic sorting using the MACS system (Milteny). 8510.sup.4 CD34+ cells were thus obtained.

(25) These purified CD34+ cells were then seeded at 510.sup.4 per ml (17 ml total) in Iscove's Modified Dulbecco's Medium (IMDM)-Glutamax (Invitrogen) supplemented with penicillin/streptomycin 100 U/ml (P/S) (Invitrogen), 1% of sodium pyruvate (Invitrogen), 1% of vitamins (Invitrogen), 1% of glutamine (Invitrogen), 2% of non-essential amino acids (Invitrogen), 1% of a commercial solution of insulin-transferrin-sodium selenite (Invitrogen) and 0.3% of bovine albumin (BSA) (PAA).

(26) Various cytokines were added to the cell suspension in order to direct the differentiation towards mast cells: SCF at 80 ng/ml (R&D), IL-6 at 50 ng/ml (R&D) and IL-3 at 1 ng/ml (R&D). The IL-3 and the IL-6 were added only during the first week of culture. The cells were then placed in an incubator at 37 C., under a humid atmosphere containing 5% CO.sub.2.

(27) The medium was renewed once or twice a week and the cells were maintained at 710.sup.5 per ml for 8 to 12 weeks until a pure population of mast cells was obtained.

(28) Unlike the usual behaviour of this type of culture, the umbilical cord cells of 9 Sep. 2009 called ROSA KIT WT have continued to proliferate in the same culture medium containing 80 ng/ml of human SCF up until now with a doubling time of 48 hours. The cells have therefore been maintained in culture since the beginning by 1/3 or 1/4 dilution in new culture medium every 3 or 4 days. The cells can be repeatedly frozen and thawed using the conventional freezing methods (decomplemented foetal calf serum (FCS) (PAA) containing 10% of DMSO (Sigma)).

(29) 2) Characterization of the Cells

(30) The identification of the ROSA KIT WT line as a mast cell line was carried out by toluidine blue staining and May-Grnwald Giemsa (MGG) staining, transmission electron microscopy study and demonstration of intracellular tryptase and of intracellular histidine decarboxylase (HDC).

(31) After MGG staining (FIG. 1), the cells of the ROSA KIT WT line appeared to be rounded with a very high nuclear-cytoplasmic ratio. In the cytoplasm, which is basophilic, the presence of numerous dark violet-coloured granulations is noted. The number and the size of the granulations varies: they are small and sparse in the cells considered to be immature; they are numerous and large in the cells considered to be mature. Their metachromasia was revealed using the toluidine blue staining technique.

(32) The morphological characterization was completed by electron microscopy observation (FIG. 1). The mast cells of the ROSA KIT WT line observed are cells which have a large nucleus, with loose chromatin suggesting that they are quite young cells. One to two nucleoli are observed in the nucleus. The cytoplasm contains ribosomes, numerous mitochondria and numerous granulations. These granulations are of the same type as those described in human mast cells in primary culture.

(33) Demonstration of the KIT Receptor and of the IgE Receptor

(34) Direct labelling at various culture times was carried out by flow cytometry on the cells of the ROSA KIT WT line in culture with an antibody directed against the alpha (a) chain of the high-affinity IgE receptor coupled to the FITC fluorochrome (Fluorescein) (Biolegend) or with the anti-human KIT (CD117) antibody coupled to APC (Biolegend). 1 ml of cells in culture at approximately 10.sup.6 cells/ml is used for each tube. The cells are centrifuged in order to remove the culture medium. The pellets are taken up in 100 l of PBS+1% FCS, and incubated for 45 min at +4 C. in the presence of anti-human IgE antibody coupled to FITC (20 l/l million cells), or in the presence of anti-human KIT antibody coupled to APC (20 l/l million cells). Two rinses are carried out in 3 ml of PBS, 0.1% FCS, and then the tubes are centrifuged. The pellets are collected in 300 l of PBS, 0.1% FCS.

(35) The reading is carried out by flow cytometry (BD FACSCalibur).

(36) As shown in FIG. 2, it appears that the cells of the ROSA KIT WT line that are analysed significantly express the IgE receptor and also the KIT receptor.

(37) Demonstration of the Expression of Tryptase and of Histidine Decarboxylase (HDC)

(38) The presence of intracellular tryptase and of intracellular histidine decarboxylase (HDC) was demonstrated in the cells of the ROSA KIT WT line by immunohistochemical methods on cytospun cells.

(39) The tryptase and the HDC are detected using a monoclonal anti-tryptase antibody (Dako) or an anti-HDC antibody (Santa Cruz).

(40) Indirect labelling was carried out on slides after cytospinning of the cells for 10 minutes at 500 revolutions per minute. After cytospinning, the cells are fixed with acetone for 8 min. They are then rinsed twice in TBS and air-dried for 20 min, then incubated with the first antibody (anti-tryptase diluted to 1/250.sup.th in TBS-1% BSA, or anti-HDC diluted to 1/250) for 1 hour at ambient temperature in a humid chamber. After three rinses in TBS (35 min), the cells are incubated with the second antibody (biotinylated goat anti-mouse IgG (Dako)) for 30 min at ambient temperature in a humid chamber, and then rinsed three times (5 min each time) in TBS. The cells are then incubated with the third antibody (Streptavidin AP (Dako)) for 30 min at ambient temperature in a humid chamber. After three rinses in TBS (35 min), 100 l of New Fuchsin are applied to each slide for 10 minutes. Rinsing is again carried out three times (5 min each time) in TBS, followed by incubation at ambient temperature for 3 min with haematoxylin. Finally, the slides are rinsed for 5 min with H.sub.2O and are mounted in Aquatex.

(41) The reading is carried out under an optical microscope.

(42) Positive labelling of the two enzymes in virtually all the ROSA cells is observed (FIG. 3). The strong tryptase positivity confirms the mast cell nature of the cells of the ROSA KIT WT line.

(43) 3) Effect of the Treatment with IL-4 or with Other Molecules of the Cells of the ROSA KIT WT Line on FcRI and KIT Expression

(44) Since it has been shown in the literature that interleukin-4 (IL-4) induces a decrease in KIT expression and an increase in FcR1 expression by normal human mast cells, the inventors wanted to verify whether this phenomenon could also be observed on the ROSA KIT WT line. For this, they used the following technique: 10 ml of cells in culture at approximately 210.sup.5 cell/ml are used for each tube. The cells are centrifuged and the pellet is taken up in 10 ml of new culture medium and then the cells are treated with IL-4 (20 ng/ml) (R&D) for 5 days. The cells are centrifuged in order to remove the culture medium. The pellets are taken up in 100 l of PBS+1% FCS, and incubated for 45 min at +4 C. in the presence of anti-human IgE antibody coupled to FITC (20 l/l million cells (Merck Bioscience)), or in the presence of anti-human KIT antibody coupled to APC (20 l/l million cells) (Biolegend). Two rinses are carried out in 3 ml of PBS, 0.1% FCS, and then the tubes are centrifuged. The pellets are collected in 300 l of PBS, 0.1% FCS.

(45) The reading is carried out by FACS (BD FACSCalibur).

(46) The results obtained are given in FIG. 4 and show that this treatment with IL-4 indeed induces a decrease in KIT expression and an increase in that of the high-affinity IgE receptor on the ROSA KIT WT cells. These cells therefore indeed behave like normal human mast cells from this point of view.

(47) 4) Demonstration of the Activation of the Cells of the ROSA KIT WT Line by the IgE-Anti-IgE Pairing

(48) In order to know whether the cells of the ROSA KIT WT line can be activated by the IgE-anti-IgE antibody coupling, resulting in degranulation with immediate release of histamine and of -hexosaminidase associated with a significant increase in membrane CD203c expression and with delayed release of TNF-alpha, cells are treated with IL-4 (20 ng/ml) and IgE (2 g/ml) (Merck Bioscience) for 5 days and then stimulated with anti-IgE (5 or 10 g/ml) (Biovalley) or with the calcium ionophore (Cai) (Sigma) (1 mol/l) for 1 or 6 hours. The 1 hour supernatant and also the pellet are used to assay the histamine and to measure the expression of CD203c at the cell surface, while the TNF-alpha is measured by a specific ELISA method in the supernatant of the cells after 6 hours of stimulation.

(49) a) Increase in Membrane CD203c Expression on the Cells of the ROSA KIT WT Line Treated with IL-4 and IgE and then Stimulated with Anti-IgE

(50) One ml of cells treated with IL-4 (20 ng/ml) and IgE (2 g/ml) for 5 days are washed twice with 3 ml of PBS and then again placed in culture medium at a concentration of 110.sup.6 cells/ml. These cells are then stimulated with anti-IgE (5 or 10 g/ml) or with the calcium ionophore (Cai) (1 mol/l) for 1 hour. The cells are washed with PBS and put back into 100 l of PBS, 1% FCS in the presence of an antibody directed against human CD203c, coupled to phycoerythrin (PE) (20 l/l million cells) (Biolegend).

(51) The results, presented in FIG. 5, show a clear increase in CD203c expression when these cells are treated with IL-4+ IgE and stimulated with anti-IgE. This increase is also observed when these cells are treated with the calcium ionophore.

(52) b) Release of -Hexosaminidase into the Supernatant of the Cells of the ROSA KIT WT Line after Activation

(53) The activation of the cells of the ROSA KIT WT line was also measured by determining the released -hexosaminidase activity. ROSA cells treated with IL-4 (20 ng/ml) and IgE (2 g/ml) are subsequently activated for 1 hour at 37 C. in the presence of 5% CO.sub.2, with the anti-IgE antibody (5 g/ml) or with the calcium ionophore (Cai) (1 mol/l).

(54) The cells are then centrifuged and the supernatants are recovered and frozen at 80 C. until the time of the assay. The cells are then lysed. The -hexosaminidase activity is measured in the supernatant and also in the cell pellet as described by Schwartz and Austen, J Invest Dermatol., 74, 349-353, 1980.

(55) The hydrolysis of p-nitrophenyl-2-acetamido-2-deoxy--D-glucopyranoside (or 4-nitrophenyl-N-acetyl--D-glucosaminide) by -hexosaminidase leads to the release of a chromophore product: p-nitrophenol. The latter is measured by spectrophotometry at 405 nm. 50 l of p-nitrophenyl-2-acetamido-2-deoxy--D-glucopyranoside (Sigma) are added to 5 l of supernatant Incubation for 2 hours at 37 C. with stirring. Addition of 150 l of stop solution (7.5 g of glycine in 500 ml of H.sub.2O, pH=10.7). Reading at 405 nm.

(56) The results obtained and presented in FIG. 6 show that the stimulation of the cells of the ROSA KIT WT line by the IgE-anti-IgE pairing leads to an induction of -hexosaminidase release which can range up to 38% of the total enzyme content of the cells. As expected, the calcium ionophore, a non-specific stimulant, induces a -hexosaminidase release which can range up to 80% of the total enzyme content of the cells, which is in accordance with the data from the literature.

(57) c) Measurement of the Histamine Release by the Cells of the ROSA KIT WT Line after their Stimulation

(58) 1) Cell Preparation:

(59) 50,000 cells treated with IL-4 (20 ng/ml) and IgE (2 g/ml) for 1 or 4 days were activated for 30 minutes at 37 C. in the presence of 5% CO.sub.2 using various concentrations of the anti-IgE antibody. The supernatants and the pellets are recovered and frozen at 80 C. until the time of the histamine assay.

(60) 2) Histamine Assay:

(61) The histamine assay was carried out in the supernatants and the pellets using a radio-immunology assay kit (histamine radioimmunoassay (RIA); Immunotech, Marseille, France) in accordance with the supplier's indications (Morel and Delaage, J Allergy Clin Immunol. 1988 October, 82(4):646-54; Valent et al., Proc Natl Acad Sci USA. 1989 July, 86(14):5542-6).

(62) The net percentage of histamine release is calculated using the following formula:
% release=SS control100/(S+P)S control
in which:
S=histamine in the supernatant, and
P=histamine in the pellet.

(63) The results obtained (FIG. 7) show that there is a dose-dependent increase (as a function of the concentration of anti-IgE added) in histamine release by the cells of the ROSA KIT WT line when they are stimulated with the IgE-anti-IgE pairing. This release is all the more significant if the cells have been pre-sensitized with IL-4 and IgEs, preferably for at least 4 days. The maximum histamine release obtained, around 40% of the total histamine content of the cells, is absolutely comparable to the beta-hexosaminidase release by the same cells stimulated under the same conditions (FIG. 6).

(64) d) Assays of the TNF-Alpha Released into the Supernatant of the Cells of the ROSA KIT WT Line after their Stimulation

(65) Using a specific ELISA kit (Enzyme Linked Immunosorbent Assay, R&D), the late release of TN F-alpha by the ROSA cells treated with IL-4 (20 ng/ml) and IgE (2 g/ml), and subsequently activated for 6 hours at 37 C., in the presence of 5% CO.sub.2, with various concentrations of the anti-IgE antibody (5 or 10 g/ml) or with the calcium ionophore (Cai) (1 mol/l), was quantified.

(66) The cells are subsequently centrifuged and the supernatants are recovered and frozen at 80 C. until the time of the TNF-alpha assay.

(67) The results obtained and presented in FIG. 8 show that there is a dose-dependent increase in TNF-alpha release by the ROSA KIT WT cells after stimulation thereof with the IgE-anti-IgE pairing.

(68) 5) Demonstration of the Ability of the Cells of the ROSA KIT WT Line to Terminally Mature after Coculture on a Sublayer of MS-5 Cells

(69) The cells of the ROSA KIT WT line have a relatively immature human mast cell phenotype (chymase negativity, relatively low metachromasia). It had previously been demonstrated that the culturing of human CD34+ haematopoietic progenitors on a sublayer of mouse lipoblast cells (MS-5 line) enables the terminal mast cell differentiation of these progenitors (Arock et al. Ann N Y Acad Sci. 1994 May 28, 725: 59-68). Cells of the ROSA KIT WT line were therefore grown on a sublayer of MS-5 cells at confluence, for more than 3 months, the culture medium being 50% renewed every 3 or 4 days. After this coculturing time, the non-adherent cells of the coculture were analysed with respect to their morphological appearance after MGG staining (FIG. 9A), their metachromasia with respect to toluidine blue (FIG. 9B), their chymase content (immunocytochemistry) (FIG. 9C) and the expression of various surface markers, including KIT, FcR1, CD54 and CD203c (FIG. 9D). The analysis of these results shows that the cells of the ROSA KIT WT line can acquire a totally mature human mast cell (MCTC) phenotype after this coculturing time. Indeed, the cells exhibit a considerable increase in the number of granulations in their cytoplasm (MGG staining) which are metachromatic (toluidine blue staining). In addition, the cocultured cells express chymase and are more strongly positive than the non-cocultured cells in terms of the expression of FcR1, CD54 and CD203c, all three of which are maturation markers.

Example 2: Obtaining of Immortalized Lines of Human Mast Cells Transfected with a Kit Receptor of Abnormal Structure: The Rosa Kit D816V AND ROSA KIT Delta 417-419 insY LINES

(70) With the aim of obtaining and characterizing SCF-independent subclones of the starting ROSA line, the cells were transfected with lentiviral vectors providing a construct encoding D816V-mutated KIT or the mutated KIT Delta 417-419 insY. These two acquired structural abnormalities of the KIT receptor are frequently encountered, respectively, during systematic mastocytoses in adults (KIT D816V; Fger et al. Int Arch Allergy Immunol. 2002 February, 127(2): 110-4) or during cutaneous mastocytoses in children (KIT Delta 417-419 insY; Bodemer et al. J Invest Dermatol 2010, 30(3): 804-15). Furthermore, it has been shown that the D816V mutation is capable of inducing systematic mastocytosis in transgenic mice (Zappulla et al. J Exp Med. 2005 Dec. 19, 202(12): 1635-41).

(71) 1) Transfection Procedure Used

(72) a) Site-Directed Mutagenesis and Plasmid Vectors

(73) In order to prepare the corresponding constructs, the cDNA encoding the short isoform of human wild-type KIT was excised from the pBS-hkitWT vector by Sal I-Acc65 digestion. The cDNA fragment was subcloned into the pENTR1A vector (Invitrogen, France). The two KIT mutations (D816V-mutated KIT or the mutated KIT Delta 417-419 insY) were introduced into the pENTR1A-hkitWT vector encoding the green fluorescent protein (GFP) using a QuickChange site-directed mutagenesis KIT (Stratagene, The Netherlands) in accordance with the manufacturer's instructions. For the KIT D816V mutant, codon 816 of WT KIT (GAC) was replaced with the GTC codon. For the KIT Delta 417-419 insY mutant, the WT KIT codons 417/418/419 (ACTTACGAC) were replaced with the TAC codon.

(74) b) Bacterial Transformation for Amplification and Purification of the Plasmid DNAs Encoding the Mutated Forms of KIT

(75) 5-10 l of plasmid are added per tube of bacteria (XL10-Gold Ultracompetent Cells #200314, Stratagene) and the bacteria are subjected to a heat shock (30 minutes on ice, then 30-40 seconds at 42 C. and then 2 minutes on ice). 1 ml of medium (SOC) without ampicillin is then added per tube and incubation is carried out for 30 minutes at 37 C. with shaking. The transformed bacteria are then plated out using sterile beads on a Petri dish containing LB medium+ampicillin (2 Petri dishes for each plasmid encoding either KIT D816V or KITD 417-419 insY). These Petri dishes are then incubated at 37 C. overnight. After this incubation, one bacteria colony is picked for each plasmid and is suspended in 200 ml of LB medium+ampicillin which is incubated at 37 C. overnight. The preparation is then centrifuged for 15 minutes at 4 C. at 8000 g, the supernatant is removed, and 12 ml of RES buffer containing RNAse (NucleoBond AX), followed by 12 ml of the LYS lysis buffer, are added to the pellet and mixing is carefully carried out by inverting the tube 8 times. The mixture is incubated for 5 minutes at ambient temperature. The lysate is then passed through the NucleoBond Xtra column, which is allowed to empty by gravity. The column is rinsed with 15 ml of EQU buffer and then the filter is discarded by turning the column upside down. The column is washed with 25 ml of WASH buffer and the plasmid DNA is eluted with the ELU elution buffer. The elution buffer with the plasmid DNA is collected in a 50 ml tube and the eluted plasmid DNA is precipitated by adding isopropanol at ambient temperature for 2 minutes. Centrifugation is carried out at 15,000 g for 30 minutes at ambient temperature, the supernatant is carefully removed and 70% ethanol at ambient temperature is added to the pellets, centrifugation is carried out at 15,000 g for 5 minutes at ambient temperature, and then the ethanol is carefully and completely removed with a pipette and the pellet is left to dry at ambient temperature. The pellet is subsequently dissolved in an appropriate volume of TE buffer and the yield and the purity of the plasmid DNA are determined by UV spectrophotometry (NanoDrop). The integrity of the plasmid is confirmed by agarose electrophoresis.

(76) c) Production of Lentiviral Stocks

(77) In order to produce lentiviral stocks, 293T cells (human epithelium) were used and the lentiviral infection was carried out by means of the calcium phosphate method according to the method described in, Zufferey et al. J Virol. 1998 December, 72(12): 9873-9880.

(78) To do this, the 293T cells (510.sup.6 cells) were seeded in 75 cm.sup.2 culture flasks (T-75) and infected the following day with 8 g of Gag-pol, 3 g of a plasmid encoding the viral envelope of the vesicular stomatitis virus G (VSV-G) and 15 g of the plasmid DNA encoding either KIT D816V or KIT Delta 417-418 419 inserY. Twenty-four hours later, the culture supernatant is filtered with a 0.45 m filter (low protein-binding Durapore; Millipore). The filtered supernatant was then ultracentrifuged at 20,000 rpm for 2 h under vacuum. After centrifugation, the supernatant was removed and the pellet (virus) was taken up in 200 l of PBS. These viral stocks were aliquoted into tubes and frozen at 80 C. until their use.

(79) d) Infection of ROSA KIT WT Cells with the Lentiviral Vectors

(80) For the infection of the ROSA KIT WT cells, the cells (10.sup.6) were incubated for 1 hour in 1 ml of medium containing SCF (80 ng/ml) and 8 g/ml of polybrene (hexadimethrine bromide). The cells were then incubated for 3 hours with variable amounts of infectious particles, centrifuged, and diluted in new medium containing SCF. The infection efficiency was measured by flow cytometry (detection of the green fluorescence of GFP) 4 days later (FIG. 10).

(81) The cells were then subcultured in the same medium for 3 weeks, and the GFP-positive infected cells were then selected by cell sorting by flow cytometry and immediately grown in an SCF-free medium. They have, since then, been maintained by regular dilution (every 3 or 4 days) in new SCF-free medium.

(82) This made it possible to establish two new SCF-independent lines, ROSA KIT D816V and ROSA KIT Delta 417-419 insY, the principal characteristics of which are described below.

(83) These two lines can be used for high-throughput screening for molecules for anti-proliferative purposes, directed either against the mutated KIT molecule, or against one or another of the intracellular molecules involved in the mutated KIT signal transduction.

(84) 2) Principal Characteristics of the ROSA KIT D816V and KIT Delta 417-419 insY Lines

(85) These two lines can be easily frozen by conventional freezing techniques (see above). The two lines have a different doubling time, about 48 hours for the ROSA KIT Delta 417-419 insY line and about 72 hours for the ROSA KIT D816V line. Their morphological appearance is also different. Specifically, while, after MGG staining, the cells of the ROSA KIT Delta 417-419 insY line appear to be homogeneous and relatively immature with few granules, the cells of the ROSA KIT D816V line appear to be more mature and more granular (FIG. 11). This is in agreement with the data from the literature showing that the introduction of the KIT D816V mutation into a mouse haematopoietic precursor line induces the appearance of mast cell differentiation characteristics (Mayerhofer et al. J Immunol. 2008 Apr. 15, 180(8): 5466-76).

(86) Moreover, the expression of certain membrane markers present on the cells of the ROSA KIT Delta 417-419 insY and KIT D816V lines, optionally present in the presence of SCF, was studied by flow cytometry, this expression being compared with that of the cells of the ROSA KIT WT line. The results obtained (presented in FIG. 12) were the following: the cells of the ROSA KIT Delta 417-419 insY and KIT D816V lines spontaneously express (in the absence of SCF) more CD38, CD203c and KIT than the cells of the ROSA KIT WT line, this increase in expression being completely abolished in the presence of SCF, the cells of the KIT D816V line express more CD54 than the cells of the other two lines, whether in the absence or in the presence of SCF, the cells of the KIT D816V line express more FcR1 than the cells of the other two lines in the absence of SCF, this phenomenon being abolished by adding SCF, the cells of the ROSA KIT Delta 417-419 insY and KIT D816V lines express, in the presence of SCF, more CD63 than the cells of the ROSA KIT WT line, this increase in expression being completely abolished in the absence of SCF, the expression of CD53 and of FcR1 is increased in the 3 lines by treatment with IL-4 (20 ng/ml), and the expression of KIT is decreased in the three lines by treatment with IL-4 (20 ng/ml).

(87) Furthermore, the inventors have studied the cloning capacity in semi-solid medium (methylcellulose) of the two lines, ROSA KIT Delta 417-419 insY and KIT D816V, in the absence of SCF, comparing it with that of the cells of the ROSA KIT WT line (in the presence of SCF at 80 ng/ml). To do this, the cultures were seeded in a proportion of 5000 per Petri dish containing 1 ml of culture medium supplemented with 1% of methylcellulose (final concentration) and incubated at 37 C. in a humid atmosphere containing 5% CO.sub.2 in air. The colonies were counted after 28 days of incubation for ROSA KIT WT and 15 days of incubation for ROSA KIT Delta 417-419 insY and KIT D816V. An example of culturing in semi-solid medium is shown in FIG. 12 and the results obtained are presented in Table I.

(88) TABLE-US-00001 TABLE I Study of the clonogenicity of the cells of the ROSA KIT WT, KIT Delta 417-419 insY and KIT D816V lines in semi-solid medium (1% methylcellulose) Number Number of of Number of Percentage of colonies colonies colonies clonogenic Line dish 1 dish 2 dish 3 Mean cells ROSA KIT WT 238 408 372 333 6.8% ROSA KIT 173 438 445 352 7.04% Delta 417-419 insY ROSA KIT 203 117 292 204 4.08% D816V

(89) The results of these experiments show that the cells of the ROSA KIT WT and KIT Delta 417-419 insY lines have a comparable clonogenicity of about 7%, whereas the cells of the ROSA KIT D816V line are less clonogenic (percentage of clonogenic cells around 4%), which is in agreement with the slightly longer doubling time and the more mature appearance of the cells of the ROSA KIT D816V line.

(90) Moreover, the inventors analysed the structure of the KIT receptor in the 3 ROSA KIT WT, ROSA KIT Delta 417-419 insY and ROSA KIT D816V lines. To do so, the total RNA was extracted from the cells of each line using an RNeasy Mini kit (Qiagen SA, Courtaboeuf, France). The RNA was reverse transcribed to cDNA using a StrataScript first strand synthesis system (Stratagene, Massy, France) and random hexamer primers in a total volume of 25 ml, according to the manufacturer's instructions. The KIT coding sequences were then amplified by PCR from 2.5 ml of cDNA, using HotStarTaq DNA polymerase (Qiagen SA) and the primers already published (Bodemer C et al., J Invest Dermatol. 2010 March, 130(3): 804-815), by applying 40 cycles at 94 C. for 30 seconds, 57 C. for 30 seconds, and 72 C. for 45 seconds. The PCR products were purified using a GeneClean III kit (Qbiogene, Illkirch-Graffenstaden, France), and all the KIT coding regions were directly sequenced using a BigDye Terminator v1.1 kit (Applied Biosystems, Courtaboeuf, France), the published sequencing primers (see above, PCR primers) and an ABI Prism 3100 sequencer (Applied Biosystems).

(91) The results obtained confirm the presence of wild-type KIT in the 3 ROSA KIT WT, ROSA KIT Delta 417-419 insY and ROSA KIT D816V lines. In addition, for the ROSA D816V line, the KIT sequencing shows that codon 816 of WT KIT (GAC) has been replaced with GTC. For the ROSA KIT Delta 417-419 insY line, the KIT sequencing shows that codons 417/418/419 of WT KIT (ACTTACGAC) have been replaced with the TAC codon. These results show that the ROSA KIT WT line indeed exhibits only a wild-type KIT, explaining its dependence with respect to SCF for its growth, whereas the expected KIT structure is found in the ROSA KIT Delta 417-419 insY and ROSA KIT D816V lines, which could explain their independence with respect to SCF for their proliferation.

(92) The presence of KIT and its phosphorylation state were then verified, using the Western blotting method, in the three ROSA KIT WT, ROSA KIT Delta 417-419 insY and ROSA KIT D816V lines, treated or not treated with recombinant human SCF (80 ng/ml), in comparison with the HMC-1.2 line. The results of these experiments, presented in FIG. 14, show that the KIT protein is spontaneously present in the 4 lines. Furthermore, there is a constitutive phosphorylation of KIT in the HMC-1.2 line, and also in the ROSA KIT Delta 417-419 insY and ROSA KIT D816V lines, this being the case in the absence of SCF. On the other hand, KIT is phosphorylated on tyrosine only in the presence of SCF. These results confirm that the independence with respect to SCF of the ROSA KIT Delta 417-419 insY and ROSA KIT D816V lines is indeed due to a constitutive phosphorylation of the mutated KIT receptor in these two lines.

(93) Finally, the inventors evaluated the effect of two molecules which inhibit tyrosine kinase activity, imatinib and dasatinib (both supplied by Sequoia Research), on the proliferation of the 3 ROSA KIT WT, ROSA KIT D816V and ROSA KIT Delta 417-419 insY lines. To do this, the cells were seeded at a starting concentration of 310.sup.5 cells per ml in 96-well plates (100 l per well) and incubated at 37 C. in an incubator (5% CO.sub.2 in air) for 48 hours in the presence of 1 M/l of imatinib or of dasatinib (in DMSO brought to a final concentration of 0.1%) or in the presence of DMSO alone (0.1% final concentration) in their usual culture medium (containing human SCF at 80 ng/ml for the ROSA KIT WT line but without SCF for the ROSA KIT D816V and ROSA KIT Delta 417-419 insY lines). At the end of this incubation, 10 l of WST-1 (Roche Applied Science) are added to each well and the cells are incubated for a further 3 hours in an incubator at 37 C. The number of live cells is then measured for each condition by reading the absorbance at 450 nm using a Multiskan MS plate reader (Thermo LabSystems).

(94) The results obtained for each line (presented in FIG. 15) are expressed as % of live cells (the condition DMSO alone at 0.1% final concentration being considered to contain 100% of live cells).

(95) The results obtained are completely in accordance with those of the literature (Shah et al. Blood. 2006 Jul. 1, 108(1): 286-91). Specifically, they show that both the cells of the ROSA KIT WT line and those of the ROSA KIT Delta 417-419 insY line are sensitive to the inhibitory effect of Imatinib (inhibitor of WT KIT or of mutated KIT at the level of the extracellular domain of KIT) and to the inhibitory effect of dasatinib. On the other hand, and as expected, imatinib is incapable of inhibiting the proliferation of the cells of the ROSA KIT D816V line.

(96) These results thus show that these three cell lines are perfectly suited to differential screening for inhibitors of the various forms of wild-type KIT or of mutated KIT.

REFERENCES

(97) 1. Razin E, Ihle J N, Seldin D, Mencia-Huerta J M, Katz H R, LeBlanc P A, Hein A, Caulfield J P, Austen K F, Stevens R L. Interleukin 3: A differentiation and growth factor for the mouse mast cell that contains chondroitin sulfate E proteoglycan. J Immunol. 1984 March; 132(3): 1479-86. 2. Levi-Schaffer F, Austen K F, Gravallese P M, Stevens R L. Coculture of interleukin 3-dependent mouse mast cells with fibroblasts results in a phenotypic change of the mast cells. Proc Natl Acad Sci USA. 1986 September; 83(17): 6485-8. 3. Tadokoro K, Stadler B M, De Weck A L. Factor-dependent in vitro growth of human normal bone marrow-derived basophil-like cells. J Exp Med. 1983 Sep. 1; 158(3): 857-71. 4. Moqbel R, Wakelin D, MacDonald A J, King S J, Grencis R K, Kay A B. Release of leukotrienes during rapid expulsion of Trichinella spiralis from immune rats. Immunology. 1987 March; 60(3): 425-30. 5. Dayton E T, Pharr P, Ogawa M, Serafin W E, Austen K F, Levi-Schaffer F, Stevens R L. 3T3 fibroblasts induce cloned interleukin 3-dependent mouse mast cells to resemble connective tissue mast cells in granular constituency. Proc Natl Acad Sci USA. 1988 January; 85(2): 569-72. 6. Butterfield J H, Weiler D, Dewald G, Gleich G J. Establishment of an immature mast cell line from a patient with mast cell leukaemia. Leuk Res. 1988; 12(4): 345-55. 7. Befus A D. Mast cells are that polymorphic! Reg Immunol. 1989 May-June; 2(3): 176-87. 8. Burd P R, Rogers H W, Gordon J R, Martin C A, Jayaraman S, Wilson S D, Dvorak A M, Galli S J, Dorf M E. Interleukin 3-dependent and -independent mast cells stimulated with IgE and antigen express multiple cytokines. J Exp Med. 1989 Jul. 1; 170(1): 245-57. 9. Furitsu T, Saito H, Dvorak A M, Schwartz L B, Irani A M, Burdick J F, Ishizaka K, Ishizaka T. Development of human mast cells in vitro. Proc Natl Acad Sci USA. 1989 December; 86(24): 10039-43. 10. Galli S J. New insights into the riddle of the mast cells: microenvironmental regulation of mast cell development and phenotypic heterogeneity. Lab Invest. 1990 January; 62(1): 5-33. 11. Schaeffer W I. Terminology associated with cell, tissue, and organ culture, molecular biology, and molecular genetics. Tissue Culture Association Terminology Committee. In Vitro Cell Dev Biol. 1990 January; 26(1): 97-101. 12. Furitsu T, Tsujimura T, Tono T, Ikeda H, Kitayama H, Koshimizu U, Sugahara H, Butterfield J H, Ashman L K, Kanayama Y. Identification of mutations in the coding sequence of the proto-oncogene c-kit in a human mast cell leukaemia cell line causing ligand-independent activation of c-kit product. J Clin Invest. 1993 October; 92(4): 1736-44. 13. Valent P. The riddle of the mast cell: kit(CD117)-ligand as the missing link? Immunol Today. 1994 March; 15(3): 111-4. 14. Abraham S N, Malaviya R. Mast cells in infection and immunity. Infect Immun. 1997 September; 65(9): 3501-8. 15. Kirshenbaum A S, Goff J P, Semere T, Foster B, Scott L M, Metcalfe D D. Demonstration that human mast cells arise from a progenitor cell population that is CD34(+), c-kit(+), and expresses aminopeptidase N (CD13). Blood. 1999 Oct. 1; 94(7): 2333-42. 16. Varadaradjalou S, Fger F, Thieblemont N, Hamouda N B, Pleau J M, Dy M, Arock M. Toll-like receptor 2 (TLR2) and TLR4 differentially activate human mast cells. Eur J Immunol. 2003 April; 33(4): 899-906. 17. Kirshenbaum A S, Akin C, Wu Y, Rottem M, Goff J P, Beaven M A, Rao V K, Metcalfe D D. Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukaemia; activation following aggregation of FcepsilonR1 or FcgammaRI. Leuk Res. 2003; 27(8): 677-82. 18. Yoshikubo T, Inoue T, Noguchi M, Okabe H. Differentiation and maintenance of mast cells from CD34(+) human cord blood cells. Exp Hematol. 2006; 34(3): 320-9. 19. Valent P, Akin C, Escribano L, Fdinger M, Hartmann K, Brockow K, Castells M, Sperr W R, Kluin-Nelemans H C, Hamdy N A, Lortholary O, Robyn J, van Doormaal J, Sotlar K, Hauswirth A W, Arock M, Hermine O, Hellmann A, Triggiani M, Niedoszytko M, Schwartz L B, Orfao A, Horny H P, Metcalfe D D. Standards and standardization in mastocytosis: consensus statements on diagnostics, treatment recommendations and response criteria. Eur J Clin Invest. 2007; 37(6): 435-53. 20. Malbec O, Roget K, Schiffer C, Iannascoli B, Dumas A R, Arock M, Daron M. Peritoneal cell-derived mast cells: an in vitro model of mature serosal-type mouse mast cells. J Immunol. 2007; 178(10): 6465-75. 21. Mayerhofer M, Gleixner K V, Hoelbl A, Florian S, Hoermann G, Aichberger K J, Bilban M, Esterbauer H, Krauth M T, Sperr W R, Longley J B, Kralovics R, Moriggl R, Zappulla J, Liblau R S, Schwarzinger I, Sexl V, Sillaber C, Valent P. Unique effects of KIT D816V in BaF3 cells: induction of cluster formation, histamine synthesis, and early mast cell differentiation antigens. J Immunol. 2008 Apr. 15; 180(8): 5466-76. 22. Bodemer C, Hermine O, Palmrini F, Yang Y, Grandpeix-Guyodo C, Leventhal P S, Hadj-Rabia S, Nasca L, Georgin-Lavialle S, Cohen-Akenine A, Launay J M, Barete S, Feger F, Arock M, Catteau B, Sans B, Stalder J F, Skowron F, Thomas L, Lorette G, Plantin P, Bordigoni P, Lortholary O, de Prost Y, Moussy A, Sobol H, Dubreuil P. Pediatric mastocytosis is a clonal disease associated with D816V and other activating c-KIT mutations. J Invest Dermatol. 2010; 30(3): 804-15. 23. Laidlaw T M, Steinke J W, Tiana A M, Feng C, Xing W, Lam B K, Paruchuri S, Boyce J A, Borish L. Characterization of a novel human mast cell line that responds to stem cell factor and expresses functional FceRI. J Allergy Clin Immunol. 2011; 127(3): 815-22.