FR2753203A1 - Canine T-lymphocyte cell line - Google Patents
Canine T-lymphocyte cell line Download PDFInfo
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Abstract
Description
DESCRIFIION
Indication du domaine technique
La présente invention concerne l'établissement d'une lignée de cellules issues d'un lymphome cutané du chien caractérisée en ce que cette lignée pousse de façon continue et produit des particules virales associées à une activité enzymatique de transcriptase inverse.DESCRIFIION
Technical field indication
The present invention relates to the establishment of a cell line derived from a cutaneous lymphoma of the dog, characterized in that this line grows continuously and produces viral particles associated with an enzymatic activity of reverse transcriptase.
Indication de l'état de l'art
Dans la majorité des espèces animales et chez l'homme, de nombreux rétrovirus endogènes ou exogènes ont été isolés et associés à de nombreuses pathologies.State of the art indication
In the majority of animal species and in humans, many endogenous or exogenous retroviruses have been isolated and associated with many pathologies.
Malgré les efforts en ce sens, aucun rétrovirus n'a pu être isolé chez le chien.Despite efforts in this direction, no retrovirus has been isolated in dogs.
Cependant, l'activité transcriptase inverse a été recemment mise en évidence dans un cas d'immunodépression [Modiano, JF et al., J. Comp. Path. 1995, 112: 165-183]
Dans un cas de leucémie canine, des particules rétrovirales ont été mises en évidence en microscopie électronique [Safran, N. Perk, K, Eyal, O. Res. Vet.However, reverse transcriptase activity has recently been demonstrated in a case of immunosuppression [Modiano, JF et al., J. Comp. Path. 1995, 112: 165-183]
In a case of canine leukemia, retroviral particles have been detected by electron microscopy [Safran, N. Perk, K, Eyal, O. Res. Vet.
Sci. ,1992, 52 : 250-255]. Ces divers travaux n'ont pas permis d'isoler et d'identifier la nature de ce rétrovirus. En effet, dans chaque cas, les auteurs n'ont pas réussi à établir une lignée cellulaire continue.Sci. , 1992, 52: 250-255]. These various studies have not made it possible to isolate and identify the nature of this retrovirus. In fact, in each case, the authors failed to establish a continuous cell line.
Historique de l'invention
Nous avons étudié un chien de 13 ans atteint d'un lymphome cutanéo-muqueux avec infiltration sanguine. Cette forme particulière est couramment appelée syndrome de Sézary. Cette leucémie est caractérisée par la présence de grands lymphocytes à cytoplasme granuleux et d'immunophénotype CD8+, infiltrant simultanément la peau et les ganglions. Une lignée cellulaire continue a pu être obtenue dans notre laboratoire à partir d'un ganglion tumoral. L'étude ultrastructurale de cette lignée a révélé la présence de particules rétro-virales de type
A dans le cytoplasme des cellules tumorales et de type C libérées dans le milieu extérieur. Ces particules rétrovirales appartiennent à la famille des oncovirus. History of the invention
We studied a 13-year-old dog with cutaneous and mucous lymphoma with blood infiltration. This particular form is commonly called Sézary syndrome. This leukemia is characterized by the presence of large lymphocytes with granular cytoplasm and of CD8 + immunophenotype, simultaneously infiltrating the skin and the lymph nodes. A continuous cell line could be obtained in our laboratory from a tumor node. The ultrastructural study of this line revealed the presence of retro-viral particles of the type
A in the cytoplasm of tumor cells and of type C released in the external environment. These retroviral particles belong to the oncovirus family.
Exposé de l'invention I- Etablissement de la lignée DCL 01
1- Culture à long terme de DCL 01
Le ganglion mandibulaire prélevé stérilement est disséqué dans une boîte de Pétri, puis broyé sur une grille métallique. Les lymphocytes libérés sont récupérés dans du milieu de culture et lavés trois fois dans du RPMI 1640.Description of the invention I- Establishment of the DCL 01 line
1- Long-term culture of DCL 01
The mandibular node removed sterile is dissected in a petri dish, then ground on a metal grid. The released lymphocytes are recovered in culture medium and washed three times in RPMI 1640.
Les lymphocytes ainsi isolés sont incubés à une concentration finale de 1 million/ml dans du RPMI 1640 supplémenté de 20% de sérum de veau foetal décomplémenté, de 1 mM de pyruvate de sodium, de 2 mM de L- glutamine, de 0,2 mI de pimaricine, de 100 UI de pénicilline et 100 mg de streptomycine. Les cellules sont maintenues à 37 dans une étuve contenant 5% de C02. Le milieu de culture est renouvelé tous les deux jours. Après 3 semaines de culture, des grappes de cellules lymphoïdes sont libérées dans le surnageant, à partir desquelles peut être effectué le repiquage de la culture. Après deux mois de culture, une lignés continue est obtenue et les cellules sont congelées à raison de 107 cellules par ml dans du sérum de veau foetal contenant 10% de diméthylsulfoxide (DMSO). Après plusieurs passages, les cellules sont dédoublées tous les jours et réensemencées à raison de 5.106 par ml. Le pourcentage de SVF utilisé dans le milieu de culture est rapidement diminué à 10%, puis 5%. La croissance cellulaire reste toujours meilleure en plaque 24 puits. Cependant, après plusieurs passages, les cellules peuvent se multiplier correctement en flacon Falcon.The lymphocytes thus isolated are incubated at a final concentration of 1 million / ml in RPMI 1640 supplemented with 20% of decomplemented fetal calf serum, 1 mM of sodium pyruvate, 2 mM of L-glutamine, of 0.2 ml of pimaricin, 100 IU of penicillin and 100 mg of streptomycin. The cells are kept at 37 in an oven containing 5% CO 2. The culture medium is renewed every two days. After 3 weeks of culture, clusters of lymphoid cells are released into the supernatant, from which the culture can be subcultured. After two months of culture, a continuous line is obtained and the cells are frozen at the rate of 107 cells per ml in fetal calf serum containing 10% of dimethylsulfoxide (DMSO). After several passages, the cells are split every day and reseeded at the rate of 5.106 per ml. The percentage of FCS used in the culture medium is rapidly reduced to 10%, then 5%. Cell growth is always better in 24-well plates. However, after several passages, the cells can multiply correctly in a Falcon bottle.
2- Cinétique de croissance de DCL 01
La numération des cellules est déterminée à l'aide du test d'exclusion au bleu trypan (10 ml de la suspension sont dilués dans 90 ml de bleu trypan dilué au 1/4).2- Growth kinetics of DCL 01
The cell count is determined using the trypan blue exclusion test (10 ml of the suspension are diluted in 90 ml of trypan blue diluted 1/4).
Les cellules sont ainsi numérées tous les jours pendant une semaine. La courbe de croissance est représentée sur la figure 1.The cells are thus scanned daily for a week. The growth curve is shown in Figure 1.
Greffe des cellules à des souris SCID et Nude
Le pouvoir de prolifération et d'infiltration des cellules tumorales a pu être apprécié par injection en sous cutané ou en intra-péritonéale de 40.106 cellules à des souris SCID. Neuf jours après la greffe, de nombreux nodules cutanés apparaissent et correspondent aux différents points d'inoculation. Une ascite est également présente chez la souris dont la greffe est réalisée par voie intrapéritonéale. Les souris sont alors sacrifiées et différents organes d'aspect pathologiques sont prélevés. Une suspension cellulaire est réalisée à partir des nodules cutanés et péritonéaux ainsi qu'à partir de la rate. De nouvelles lignées ont pu être obtenues à partir de ces différents prélèvements.Cell transplant to SCID and Nude mice
The power of proliferation and infiltration of tumor cells could be appreciated by injection subcutaneously or intraperitoneally of 40.106 cells into SCID mice. Nine days after the transplant, numerous skin nodules appear and correspond to the different inoculation points. Ascites is also present in mice whose transplant is performed intraperitoneally. The mice are then sacrificed and various organs of pathological appearance are removed. A cell suspension is produced from the skin and peritoneal nodules as well as from the spleen. New lines could be obtained from these different samples.
3- Infection de lymphocytes avec le surnageant de DCL 01
Une infection des lymphocytes du sang periphérique a été obtenue avec du surnageant de culture de DCL 01. Les cellules normales infectées ont pu être immortalisées et maintenues en culture durant 3 mois.3- Infection of lymphocytes with DCL 01 supernatant
Peripheral blood lymphocyte infection was obtained with DCL 01 culture supernatant. Normal infected cells could be immortalized and kept in culture for 3 months.
L'injection des cellules productrices du virus provoque chez la souris une réponse anticorps nette avec présence d'anticorps spécifiques des bandes du rétrovirus.The injection of the virus-producing cells causes a clear antibody response in the mouse with the presence of antibodies specific for the bands of the retrovirus.
II - Caractérisation de la lignée DCLO1
1- Cytologie : microscopie optique
Les cellules d'origine, à savoir avant mise en culture, présentent un noyau à chromatine fine à aspect peigné et parfois légèrement plicaturé, non nucléolé. I1 existe des renforcements chromatiniens irréguliers. Les cytoplasmes sont étendus clairs et présentent de grosses granulations azurophiles. Après plusieurs passages en culture, les cellules perdent leurs granulations. Leur cytoplasme devient hyperbasophile et présente quelques fois des vacuolisations. Ces différentes modifications sont des critères de malignité.II - Characterization of the DCLO1 line
1- Cytology: optical microscopy
The original cells, that is to say before culturing, have a fine chromatin nucleus with a combed appearance and sometimes slightly plicated, not nucleated. There are irregular chromatin reinforcements. The cytoplasms are large, clear and have large azurophilic granulations. After several passages in culture, the cells lose their granulations. Their cytoplasm becomes hyperbasophilic and sometimes presents vacuolizations. These different modifications are criteria of malignancy.
2- Etude ultrastructurale : microscopie électronique
L'étude ultrastructurale de la lignée a permis de mettre en évidence de nombreuses particules rétrovirales de type A, majoritairement dans le réticulum endoplasmique, mais également en position périnucléaire. De très nombreux ribosomes sont répartis dans tout le cytoplasme et sont le signe d'une importante synthèse protéique. Après 30 passages, de rares bourgeons sont apparus sur la membrane des cellules tumorales. Après 4 jours de traitement à l'aide du DMSO, les cellules présentent de nombreux bourgeons et libèrent des virions matures. Le core est central et est séparé de l'enveloppe par un espace clair aux électrons. La morphologie des particules rétro-virales matures sont typiques de particules de type
C mesurant de 100 à 120 nm.2- Ultrastructural study: electron microscopy
The ultrastructural study of the line has made it possible to highlight many type A retroviral particles, mainly in the endoplasmic reticulum, but also in the perinuclear position. Numerous ribosomes are distributed throughout the cytoplasm and are the sign of an important protein synthesis. After 30 passages, rare buds appeared on the membrane of tumor cells. After 4 days of treatment with DMSO, the cells have numerous buds and release mature virions. The core is central and is separated from the envelope by a clear space for the electrons. The morphology of mature retro-viral particles are typical of type particles
C measuring from 100 to 120 nm.
3- Analyse du caryotype
Le caryotype des cellules lymphoblastoïdes a été obtenu par les techniques cytogénétiques standards : après synchronisation 17 heures en présence de FRDU et de thymidine, puis blocage des mitoses par de la colchicine, le culot cellulaire est étalé sur lame sèche et dénaturé par la chaleur (bande RHG). Le nombre de chromosomes varie de 72 à 96 pour la même lignée. L'analyse du caryotype permet de confirmer l'origine canine de la lignée.3- Analysis of the karyotype
The karyotype of lymphoblastoid cells was obtained by standard cytogenetic techniques: after synchronization for 17 hours in the presence of FRDU and thymidine, then blocking of mitoses by colchicine, the cell pellet is spread on a dry slide and denatured by heat (strip RHG). The number of chromosomes varies from 72 to 96 for the same line. Karyotype analysis confirms the canine origin of the line.
On a pu noter la présence de nombreux chromosomes minutes caractéristiques de la malignité de la lignée DCL 01
4- Etude immunophénotypique : cytométrie en flux
Avant mise en culture, les cellules tumorales sont CD3+, CD8+, CD5+ et CD45+.We could note the presence of many minute chromosomes characteristic of the malignancy of the DCL 01 line.
4- Immunophenotypic study: flow cytometry
Before cultivation, the tumor cells are CD3 +, CD8 +, CD5 + and CD45 +.
Une étude immunophénotypique en cytométrie en flux de la lignée a montré une perte des antigènes membranaires (CD8-, CD5- et CD45-) avec une faible expression de l'antigène CD3 (chaîne e intracytoplasmique). Les anticorps utilisés sont spécifiques de l'espèce canine, à l'exception du CD3 qui est un anticorps polyclonal humain présentant une réactivité croisée entre l'homme et le chien. Compte tenu de cette dédifférenciation, les cellules sont préalablement stimulées par des agents connus pour induire la différenciation des cellules souches ou pour permettre la réexpression des antigènes.An immunophenotypic study in flow cytometry of the line showed a loss of membrane antigens (CD8-, CD5- and CD45-) with a low expression of the CD3 antigen (intracytoplasmic chain e). The antibodies used are specific to the canine species, with the exception of CD3 which is a human polyclonal antibody exhibiting cross-reactivity between humans and dogs. Given this dedifferentiation, the cells are previously stimulated by agents known to induce the differentiation of stem cells or to allow the re-expression of antigens.
Les cellules sont incubées pendant 3 jours avec 0,5% de DMSO et/ou 20 nmol de phorbol 12-myristate 13-acétate (PMA). Les cellules stimulées réexpriment les antigènes de membrane CD45, CD8 et DQ. La lignée cellulaire ainsi traitée présente un phénotype similaire aux cellules d'origine.The cells are incubated for 3 days with 0.5% DMSO and / or 20 nmol of phorbol 12-myristate 13-acetate (PMA). The stimulated cells re-express the CD45, CD8 and DQ membrane antigens. The cell line thus treated has a phenotype similar to the original cells.
III Analyse des protéines dans le surnageant de culture de DCL 01:
1- Electrophorèse en gel de polyacrylamide:
Les surnageants de culture et du milieu de culture (milieu utilisé pour l'établissement de la lignée) sont préalablement concentrés dans une membrane de dialyse. Le dosage des protéines est effectué selon la technique de Lowry. Il apparaît une quantité supérieure de protéines dans le surnageant de culture (146 mg/ml) en comparaison avec le milieu de culture (120 mg/ml). Les solutions d'échantillons sont déposées à la surface du gel de concentration (à 4%) à raison de 150 et 200 mg de protéines par cm de gel. Cette migration s'effectue à ampérage constant, pendant 1 heure à 20 mA pour le gel de concentration puis 2 heures à 40 mA pour le gel de séparation à 10%. Une fois l'électrophorèse terminée, un transfert électrophorétique des protéines est réalisé sur une feuille de nitrocellulose. Les protéines sont ensuite colorées au rouge Ponceau. La feuille de nitrocellulose, préalablement saturée en
PBS-lait, est découpée en petites bandelettes pour la réalisation des immunoblots. III Analysis of the proteins in the culture supernatant of DCL 01:
1- Polyacrylamide gel electrophoresis:
The culture supernatants and the culture medium (medium used for establishing the line) are previously concentrated in a dialysis membrane. The protein assay is carried out according to the Lowry technique. A higher amount of protein appears in the culture supernatant (146 mg / ml) in comparison with the culture medium (120 mg / ml). The sample solutions are deposited on the surface of the concentration gel (at 4%) at the rate of 150 and 200 mg of proteins per cm of gel. This migration is carried out at constant amperage, for 1 hour at 20 mA for the concentration gel and then 2 hours at 40 mA for the 10% separation gel. Once the electrophoresis is complete, an electrophoretic transfer of the proteins is carried out on a nitrocellulose sheet. The proteins are then stained with Ponceau red. The nitrocellulose sheet, previously saturated with
PBS-lait, is cut into small strips for the production of immunoblots.
2- Mise en évidence par la technique d'immunoblot des antigènes du surnageant reconnus par des anticorps présents chez des chiens malades:
Chaque bandelette est placée dans une rigole . Les différents sérums (chien malade et chien témoin) dilués au 1/100 dans du PBS-Tween-lait, sont mis à incuber pendant 2 heures sous agitation. Après trois lavages en PBS-Tween, les bandelettes sont mises en contact pendant 1 heure 1/2 avec l'anti-immunoglobuline canine couplée à la peroxydase. La réaction est révélée par incubation des bandelettes, à la température du laboratoire, pendant 15 minutes avec le substrat. Les résultats de l'immunoblot figurent sur la figure 2. Compte tenu du fort taux de protéines dans le surnageant, il apparaît après marquage de très nombreuses bandes dont certaines sont très denses. Il est donc difficile par cette technique d'identifier des bandes discrètes ou masquées par de plus grandes bandes. Cependant trois bandes sont clairement marquées par le sérum du chien leucémique et ne sont pas reconnues par le sérum témoin. Les poids moléculaires des trois protéines identifiées sont de ,49 kDa et 32 kDa. On peut donc purifier ces antigènes viraux protéiques de 49000 et 32000 daltons utilisables dans l'élaboration de vaccins.2- Demonstration by the immunoblot technique of the supernatant antigens recognized by antibodies present in sick dogs:
Each strip is placed in a channel. The different sera (sick dog and control dog) diluted 1/100 in PBS-Tween-milk, are incubated for 2 hours with shaking. After three washes in PBS-Tween, the strips are brought into contact for 1 hour and a half with canine anti-immunoglobulin coupled to peroxidase. The reaction is revealed by incubating the strips, at laboratory temperature, for 15 minutes with the substrate. The results of the immunoblot are shown in Figure 2. Given the high protein level in the supernatant, it appears after labeling very many bands, some of which are very dense. It is therefore difficult with this technique to identify discrete bands or masked by larger bands. However, three bands are clearly marked by the serum of the leukemic dog and are not recognized by the control serum. The molecular weights of the three proteins identified are, 49 kDa and 32 kDa. It is therefore possible to purify these protein viral antigens of 49,000 and 32,000 daltons which can be used in the development of vaccines.
Présence d'une activité transcriptase inverse.Presence of reverse transcriptase activity.
Une activité Reverse Transcriptase (RT) dépendante du manganèse a été retrouvée dans le surnageant de culture de DCL 01 selon la technique utilisée par Gross. S.,
Traktnan P. et Baltimore D. g Virol. 1981, 38 : 239-248] modifiée. Manganese-dependent reverse transcriptase (RT) activity was found in the DCL 01 culture supernatant according to the technique used by Gross. S.,
Traktnan P. and Baltimore D. g Virol. 1981, 38: 239-248] modified.
Claims (8)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1061129A1 (en) * | 1999-05-18 | 2000-12-20 | Etablissement de Transfusion Sanguine de Lyon | Infectious retroviruses from a leukemic dog cell line with extensive homologies to murine leukemia viruses |
| WO2000070024A3 (en) * | 1999-05-17 | 2001-05-25 | Francais Du Sang Ets | Infectious retroviruses from a leukemic dog cell line with extensive homologies to murine leukemia viruses |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5112756A (en) * | 1986-06-02 | 1992-05-12 | Canadian Patents And Development Limited | Continuous production of bovine Maedi-Visna-like viral antigens in Cf2Th cells |
| WO1995023218A1 (en) * | 1994-02-25 | 1995-08-31 | Pfizer, Inc. | Immortalized canine cells and uses therefor |
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1996
- 1996-09-10 FR FR9611231A patent/FR2753203B1/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5112756A (en) * | 1986-06-02 | 1992-05-12 | Canadian Patents And Development Limited | Continuous production of bovine Maedi-Visna-like viral antigens in Cf2Th cells |
| WO1995023218A1 (en) * | 1994-02-25 | 1995-08-31 | Pfizer, Inc. | Immortalized canine cells and uses therefor |
Non-Patent Citations (2)
| Title |
|---|
| J.F. MODIANO ET AL.: "RETROVIRUS-LIKE ACTIVITY IN AN IMMUNOSUPPRESSED DOG: PATHOLOGICAL AND IMMUNOLOGICAL FINDINGS.", JOURNAL OF COMPARATIVE PATHOLOGY, vol. 112, no. 2, February 1995 (1995-02-01), EDINBURGH, GB, pages 165 - 183, XP000674291 * |
| N. SAFRAN ET AL.: "ISOLATION AND PRELIMINARY CHARACTERISATION OF A NOVEL RETROVIRUS ISOLATED FROM A LEUKAEMIC DOG.", RESEARCH IN VETERINARY SCIENCE, vol. 52, no. 2, March 1992 (1992-03-01), OXFORD, GB, pages 250 - 255, XP000673955 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2000070024A3 (en) * | 1999-05-17 | 2001-05-25 | Francais Du Sang Ets | Infectious retroviruses from a leukemic dog cell line with extensive homologies to murine leukemia viruses |
| EP1061129A1 (en) * | 1999-05-18 | 2000-12-20 | Etablissement de Transfusion Sanguine de Lyon | Infectious retroviruses from a leukemic dog cell line with extensive homologies to murine leukemia viruses |
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