Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6467—Granzymes, e.g. granzyme A (3.4.21.78); granzyme B (3.4.21.79)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes

Definitions

• the invention relates to means for the in vitro diagnosis of constituents of cytotoxic cells and "helper" T cells which play a role in the immune response of an organism.
• the immune system is capable of responding specifically or not to the introduction into the body of viral, bacterial or allogenic antigens.
• Two types of cells are responsible for neutralizing and eliminating these different antigens: B lymphocytes and T lymphocytes. These cells provide the two forms of the immune response: the humoral response, mediated by B cells, and the cellular response, mediated by at least two cell populations, namely cytotoxic T lymphocytes (CTLs), and "natural killer” cells (NK).
• CTLs cytotoxic T lymphocytes
• NK natural killer cells
• cytolytic proteins In cell-mediated cytotoxicity, two lysis processes have been described. One involves the secretion by effector cells (CTLs and NK cells) of cytolytic proteins in the absence of second intracellular messengers.
• CTLs and NK cells effector cells
• the second process called regulated lysis, involves the storage of lytic or lysis-associated proteins in cytoplasmic granules, in large quantities, and their secretion only after activation of the surface receptors of effector cells and production of second intracellular messengers.
• various proteins have been identified, among which, the perforin or protein which forms pores, a family of proteases which are called granzymes, and proteoglycans, proteins of high molecular weight, responsible for the transport of either perforin, or granzymes, to the cell membrane.
• proteases which are called granzymes
• proteoglycans proteins of high molecular weight, responsible for the transport of either perforin, or granzymes, to the cell membrane.
• Perforin was isolated from a cytotoxic line in rats and humans.
• the work carried out made it possible to observe that in the presence of Ca 2+ , the molecules of perforin polymerize, insert themselves at the level of the plasma membrane of the target cell and form pores causing the destruction of the target cells.
• Murine and human cDNA clones have been isolated. The genomic sequences of perforin have been reported recently in mice and humans.
• granzymes As for granzymes, many teams have described their purification from CTL granules or cells with NK / LAK activity (lymphokine activated killer). They represent the majority of proteins in intracytoplasmic granules (85% versus 10% for perforin). Different granzymes have been isolated and characterized in mice as well as in humans. More particularly, six granzymes named granzymes A to F have been described in the cytoplasmic granules of murine CTLs. A seventh granzyme called granzyme G was recently characterized in mouse CTLs. In humans, two granzymes have been isolated corresponding to granzymes A and B, and a third called H does not correspond to any murine granzyme already isolated.
• the granzymes are synthesized in the form of premolecules containing a signal peptide characteristic of the secreted proteins or having a granular localization. This signal sequence is followed by an acid activating dipeptide which is generally Gly-Glu or Glu-Glu (propeptide) and must be cleaved by a dipepdityl-peptidase to release the mature protein. There is great homology between the different granzymes, which suggests that they belong to a family of granular serine proteases.
• TcR T receptor to the antigen
• the granzymes by proteolysis, could cleave constituents of the extracellular matrix or components of the interstitial medium and thus be involved in the phenomena of migration and infiltration of lymphocytes into the tissues during inflammatory processes.
• proteoglycans Other proteins have been characterized in the granules of CTLs and "NK" cells: these are proteoglycans. These molecules are said to play a role in the "packaging" of perforin and granzymes. They can also constitute a sort of protective barrier on the internal face of the secretory granules, thus protecting the cytotoxic cell from the effects of its own lytic molecules. Perforin and granzymes have been extensively studied in terms of their gene expression by the techniques of
• lymphocytic infiltrates detected by histology, phenotypically characterized by immunohistochemistry and which are nevertheless devoid of the expression of the messengers of granzymes and perforin.
• the interpretation of such results remains a question and raises many questions in relation to the state of immunization of the cells of these infiltrates, the effect of immunosuppressive therapy and the actual function of these cells. Only the monitoring of patients over time should make it possible to study the evolution of these cellular infiltrates, their fate and their relationship to the evolution of the rejection or GVH process depending on the treatment administered.
• Lymphocytic infiltrates detected by histology remain in fact the only operational criterion on which clinicians rely to confirm the clinical diagnosis, whether rejection or GVH in the case of allografts.
• the object of the invention is therefore to provide new and very specific means of in vitro diagnosis in humans of the presence of the constituents of the granules mentioned above.
• the antibodies of the invention which are directed against the constituents of cytoplasmic granules of "helper" T cells or of cytotoxic cells, more especially cytotoxic T lymphocytes and "natural killer” cells, are characterized in that they are acts as monoclonal antibodies capable of specifically recognizing an epitope of a given constituent of a human granule, in particular an epitope of human granzyme or human perforin, giving rise to an antigen / antibody type reaction.
• the term "capable of specifically recognizing an epitope” as used in the description and the claims means that the monoclonal antibodies react with the epitope in question using the immunological techniques ELISA, Western Blot or Immunoprecipitation described in the examples.
• the monoclonal anti-granzyme antibodies are in particular anti-granzyme A or anti-granzyme B or even anti-granzyme H antibodies.
• the monoclonal antibodies of the invention are further characterized by the fact that they belong to the class of IgGs and that they are directed against proteins of molecular weight of approximately 25 to 30 kDa (Granzyme B), 60 kDa (Granzyme A) or approximately 66 to 75 kDa (Perforine).
• the invention also relates to any fragment of the above monoclonal antibodies as soon as it is capable of interacting specifically with a given constituent of said granules, in particular with a granzyme or perforin.
• the monoclonal antibodies of the invention are also characterized by the fact that they are as obtained by secretion from strains of hybridomas resulting from the fusion of an immortal non-secreting myeloma cell with an antibody-producing cell directed against a given constituent of granules of human cytotoxic cells.
• the step of merging the two cell types is in particular carried out according to the most commonly used technique, namely that of Kohler and Milstein, Nature, vol 256, p. 495, 1975.
• Antibody producing cells are splenocytes. These cells are recovered after in vivo immunization of the animal with the appropriate antigen.
• the constituents of the purified granules are used and injected with an adjuvant. Particularly satisfactory results are obtained by purifying the constituents of the cytoplasmic granules according to the technique of Krahenb ⁇ hl et al. in J. Immunol, 141, 3471-77, 1988.
• the constituents of the granules are in native form or, alternatively, in recombinant form.
• a recombinant human granzyme B or a recombinant human perforin is used.
• anti-granzyme monoclonal antibody means that the antibody is as induced by the native granzyme molecule and the expression "recombinant anti-granzyme monoclonal antibody” as the monoclonal antibody is as induced by a recombinant granzyme molecule.
• the other constituents of the granules such as perforin.
• Immortal cells are non-secreting myeloma cells, which makes it possible to obtain hybridomas secreting only the immunoglobulin of the specificity of the producer cell.
• the hybridomas obtained are cultured and cloned according to the limiting dilution method.
• the hybridomas whose culture supernatants have the highest titers of antibodies in ELISA are selected.
• they are injected into animals, for convenience, mice, rats or rabbits and ascites are thus produced in large quantities.
• the hybridoma strains are maintained in culture in CO 2 incubators.
• the recovered monoclonal antibodies can be used as they are or are purified, for example by affinity column and stored by freezing, or optionally lyophilized.
• the invention relates in particular to the monoclonal antibodies against recombinant human granzyme B and recombinant human anti-perforin.
• Antibodies of this type have been deposited at the
• the invention particularly relates to the anti-human granzyme B and anti-human granzyme A monoclonal antibodies produced respectively by the clones called GRB 51D and GRA 66D, deposited in the National Collection of Culture of Microorganisms (CNCM), under the numbers 1- 1060 and 1-1059, March 12, 1991. It also describes the mouse anti-perforin monoclonal antibodies deposited at the CNCM under the number I-1058 on March 12, 1991.
• the invention also relates to the strains of hybridomas producing monoclonal antibodies defined above and more especially the clones producing high titers of antibodies measured by ELISA test.
• the hybridoma strains of the invention are characterized in that they are formed from hybrid cells resulting from the fusion of immortal cells with a cell producing antibodies directed against a given constituent of cytoplasmic granules of T-helper cells or cytotoxic cells, in particular antibodies directed against granzymes or perforin.
• This method comprises the fusion and selection steps defined above in relation to the monoclonal antibodies.
• proteins against which the monoclonal antibodies of the invention are directed are either native proteins or recombinant proteins. These recombinant proteins are new products and, as such, fall within the scope of the invention.
• amino acid sequences containing at least the active C-terminal part of the mature protein are amino acid sequences containing at least the active C-terminal part of the mature protein.
• these proteins are produced in cellular hosts, in particular in bacteria, transformed by introduction of expression vectors, in particular of plasmids, containing the gene fragments coding for the amino acid sequences. wanted.
• fragments are excised from DNA clones encoding the mature proteins and are introduced by ligation into an appropriate site of the chosen vector.
• the expressed proteins are recovered from the culture medium, after lysis of the bacteria, and purified.
• transformed cell hosts expression vectors such as plasmids, and DNA fragments as mentioned above are within the scope of the invention. This targets more specifically the recombinant proteins of perforin and granzyme and the tools used for their expression.
• the invention thus relates to a fragment of the perforin sequence comprising the C-terminal active part of human perforin, more especially the fragment as expressed by the DNA fragment Ball-Bam HI by pAR 3039. It also relates a fragment of the sequence of human granzyme B as expressed by the DNA fragment Ball-Bam MI by pAR 3038.
• the invention also describes a fragment of the perforin sequence comprising the active C-terminal part of the mature mouse perforin, more especially the fragment as expressed by the 1400 bp Smal-EcoRV DNA fragment.
• the invention relates in particular to the amino acid sequence extending from position 98 to 534 in FIG. 1. It also relates to the DNA fragment capable of coding for this amino acid sequence, the vectors containing such a DNA fragment, in particular a plasmid vector, as well as the bacteria transformed by incorporation of such vectors, in particular the transformed bacteria mPerf-PL 40 deposited at the CNCM March 12, 1991 under No. 1-1057.
• the genes of granzymes and of perforin are inducible in vitro and in vivo during cellular activation in normal or pathological situation.
• Granzymes appear as early markers of cell activation; perforin appears to be preferably a marker for the cytotoxic activity of a cell. These proteins therefore appear as functional markers of cellular and / or cytotoxic activation in humans.
• the high specificity of the monoclonal antibodies of the invention makes them particularly valuable for demonstrating the presence of such markers in a fluid or a human biological sample.
• the invention therefore also relates to the application of these monoclonal antibodies as bioreagents for the in vitro diagnosis in a fluid or biological sample of the presence of constituents of the cytoplasmic granules of T "helper" cells or of cytotoxic cells more especially of granzymes or perforin or alternatively, for example, proteoglycans or lymphotoxin-like molecules.
• the monoclonal antibodies are free. Alternatively, they are fixed on a non-immunogenic solid support.
• the sample to be tested is brought into contact with a preparation of monoclonal antibodies, or of a fragment as defined above, immobilized on a solid support, under conditions suitable for the production of an antigen-antibody complex with said constituents, in particular respectively with a granzyme or perforin when they are present in the sample, then the formation of such a complex of antigen-antibody type is demonstrated.
• said constituent more particularly granzyme or perforin, comprises a marker group. It is most generally a radioactive group.
• a second antibody is used coupled to a group whose activity can be measured such as an enzyme, such as alkaline phosphatase or a fluorescent group.
• This detection method makes it possible to reveal with great sensitivity and quickly the presence of said constituents, in particular of granzymes or of perforin in a biological sample, in particular to locate and quantify them for example at the level of biopsies of pathological tissue, or peripheral blood cells.
• the monoclonal antibodies of the invention and their fragments, free or immobilized are used to carry out diagnostics in pathologies where the course of the disease modifies the level of granzyme or perforin.
• the contribution of the monoclonal antibodies of the invention is therefore important for diagnosing the presence of activated and / or cytotoxic cells in abnormally high numbers in the affected organ, or in the peripheral blood.
• the recombinant proteins, more particularly of granzyme B and granzyme H of the invention are of great interest in use as autoantigens for recognition by autoantibodies present in autoimmune sera.
• the procedure is advantageously carried out according to the usual techniques.
• the invention also relates to a kit for the in vitro diagnosis of the presence of said constituents of the cytoplasmic granules more especially of granzyme or of perforin in a biological sample.
• an appropriate solid phase serving as a support for the assay such as a micro-titration plate
• a preparation of said constituents more particularly of granzyme or of perforin with, where appropriate, a marker group, and when the preparation does not contain a marker group, a preparation of a second antibody with a marker group,
• Monoclonal antibodies are also capable of constituting therapeutic agents of great interest and can play the role of vectors of enzymes or drugs.
• FIG. 1 the amino acid sequence of the recombinant perforin, the preparation of which is reported in the examples,
• FIG. 2 the mode of construction of HLPDE3
• FIG. 3 a partial restriction map of the plasmid pAR 3038
• FIG. 5 the mode of construction of the plasmid pAR 3039-HuP
• mice used are BALB / C mice of haplotype H-2d.
• the polyclonal antibody directed against granzyme B is as obtained according to the technique described by Krâhenb ⁇ hl (see reference above).
• the REX line is a T line immortalized by the Epstein Barr virus.
• the monoclonal antibody PAb419 is directed against the T antigen of SV40 (Harlow et al., J of Virology, 861-869, 1981). 1-2 Solutions
• MILLIEU 1 RPMI 1640 (GIBCO), 100 u / ml penicillin, 100 u / ml streptomycin (GIBCO), 2 mM glutamine (GIBCO).
• MEDIUM 2 RPMI 1640 (GIBCO), 100 u / ml penicillin, 100 u / ml streptomycin (GIBCO), 2 mM glutamine (GIBCO), 250 u / ml interleukin 2 (Roussel Uclaf, 0.7 10 6 u BRMP / sample ).
• MEDIUM 3 RPMI (GIBCO), 100 u / ml penicillin, 100 u / ml streptomycin (GIBCO), 2 mM glutamine (GIBCO), 1 mM sodium pyruvate (GIBCO), 10% inactivated fetal calf serum (GIBCO), 0.36%. glucose (A.P.).
• MIDDLE 4 Dubecco's M0D Eagle Medium (GIBCO),
• MEDIUM 5 Dubecco's MOD Eagle Medium (GIBCO), 100 u / ml penicillin, 100 u / ml streptomycin (GIBCO), 2 mM glutamine (GIBCO), 1 mM Sodium pyruvate (GIBCO), 10% inactivated fetal calf serum (GIBCO), 10% inactivated horse serum (GIBCO), 10% NCTC 135 (GIBCO), 2 mM NaOH (Prolabo), 2% Hypoxanthine (GIBCO), 2% Thymidine (GIBCO), 2% Aminopterin (GIBCO).
• RIPA 10 mM Tris pH 8.1 mM EDTA, 150 mM NaCl, 1% NP40, 1% Na deoxycholate, 0.1% SDS.
• Laemmli buffer (Laemmli, Nature 227, 680-685, 1970): 62.5 mM Tris pH 6.8, 2% SDS, 15% glycerol, 5% ⁇ -mercaptoethanol, 0.01% bromophenol blue. II - Methods
• Lymphocytes are isolated from peripheral blood by ficoll gradient (PHARMACIA). After 2 washes in medium 5, the cells are counted in Hayem solution and incubated at the rate of 3 ⁇ 10 8 cells in culture flasks (COSTAR) pretreated with inactivated SHN for 1 hour at 37 ° C. The non-adherent cells are then collected, washed in RPMI SHN 10% and fractionated by centrifugation on a discontinuous percoll gradient (PHARMACIA). 4 solutions of different percoll concentration are prepared (31%, 34%, 37% and 41%). One out of two solutions is prepared in RPMI medium (red), or in isotonic Na chloride solution (colorless), thus allowing good visualization of the interfaces.
• mice with granzymes A and B purified from LGL from peripheral blood or from NK cells. 10 ⁇ g of purified proteins (granzyme A and B) dissolved in 200 ⁇ g of Vaccicox R (109 units), adjuvant, are injected intraperitoneally into a BALB / C mouse. 21 days later, a reminder is carried out thanks to a second intravenous injection (10 ⁇ g of protein dissolved in 200 ⁇ g of physiological saline).
• the mouse is sacrificed and its spleen is removed in a sterile medium. It is diluted with a 5 ml syringe plunger in a sterile Petri dish containing 5 ml of RPMI. The splenocyte solution is then washed with 50 ml of RPMI. After centrifugation, the cell pellets are stored at room temperature.
• the NS-1 myeloma cells cultured in medium 2 are centrifuged for 7 min at 1500 rpm, then washed in 50 ml of RPMI.
• the cell pellets are resuspended in 10 ml of RPMI, and transferred to the tube containing the splenocytes according to the ratio of 1 myeloma cell to 2 splenocytes. This mixture is adjusted to 40 ml with RPMI, then centrifuged for 7 min at 1500 rpm at 20 ° C.
• the mixed cell pellet is resuspended with 1 ml of PEG (polyethylene glycol 1500, BOEHRINGER), (fusing agent), by gently turning the pipette in the tube for 1 min.
• PEG polyethylene glycol 1500, BOEHRINGER
• the PEG is diluted by adding in 1 min 1 ml of RPMI 1640 preheated to 37 ° C and then 7 ml of the same medium in 2 min to reduce the toxicity of this product. After centrifugation at 1800 rpm for 4 min without brake, the cell pellet is resuspended in 10 ml of medium (4) preheated to 37 ° C.
• Cells are distributed in sterile 96-well flat-bottom plates at the rate of 105 myeloma cells in 100 ⁇ l per well (time 0).
• the preparation of the 96-well flat-bottom plates is carried out 24 hours before the test. For this purpose, 100 ⁇ l of antigen (purified granzymes A or B), at 1 ⁇ g / ml (in
• PBS PBS
• Tween 20 are performed before testing the culture supernatants of the hybridomas. The plates are incubated for 1 hour at
• the development is carried out by adding 100 ⁇ l of a solution containing the substrate (10 ⁇ l PBS, 3% H2O2 in 12.5 ml of ABTS 180 mg / 1). The plates are read by measuring the OD at 405 nm 15 to 25 min after the addition of the substrate.
• the supernatants of 28 hybridomas designated GRB were found to be specific for granzyme B in the ELISA test (test carried out with purified protein).
• the percentage of hybridomas secreting specific antibodies is particularly satisfactory, in fact out of 300 hybridomas resulting from the fusion between the NSI myeloma cells and the splenocytes of the mouse immunized with purified granzyme B, 10% secrete specific antibodies in test ELISA. In the case of granzyme A, 5% of the hybridomas of the second fusion are specific under the same conditions.
• the hybridomas whose supernatants have the highest positivity in the ELISA test are cloned into 96-well plates with flat bottom. Several dilutions are tested. Cloning is carried out at 100 cells, 10 cells, 1 cell and 0.25 cells per well at the rate of 100 ⁇ l of medium 4 per well.
• the cells are fed 3 days after cloning with 100 ⁇ l of medium 4, then every 7 days.
• the culture supernatant of these wells is tested by ELISA on the granzymes purified on granzyme A or B and on the lyzozyme as negative control.
• Positive hybridomas are transferred to 24-well plates, then to 6-well plates and thereafter in a vial to maintain growth exponential of cells. These hybridomas are injected into the peritoneum of mice to produce ascites. They can be left in culture until cell death in order to obtain highly concentrated supernatants studied in Western blotting or in immunoprecipitation.
• hybridomas secreting antibodies recognizing granzyme B called respectively GRB98C and GRB51D and three other strains secreting antibodies recognizing granzyme A, called GRA66D, GRA382E and GRA10D are recovered. The remaining hybridomas will be cloned in order to constitute a panel of antibodies directed against human granzymes A and B.
• the culture supernatant of hybridoma to be tested is diluted 1/10 in 20 mM Tris pH 7.6, 137 mM NaCl (TBS).
• TBS Tris pH 7.6, 137 mM NaCl
• the kit strip is incubated for 15 min at room temperature with the diluted supernatant. After 3 washes in TBS, 0.1% Tween 20 (TBS-T), the strip is incubated in a solution of anti-mouse antibodies coupled to peroxidase (diluted 1/500 in TBS-T).
• the revelation is carried out by incubating the strip for 15 min at room temperature in 3 ml of a solution containing the substrate (1 chloronaphthol tablet dissolved in 10 ml of cold methanol, mixed immediately with 50 ml of TBS containing a drop of hydrogen peroxide). After 3 washes in distilled water, the strips are dried and can be interpreted.
• the immunization technique used in this study made it possible to obtain hybridomas secreting antibodies of isotype IgG.
• the antibody isotype was confirmed by the kit marketed by Amersham of mouse monoclonal antibodies.
• the monoclonal antibodies GRB98C, GRA66D, GRA382E and GRA10D have the IgG1 isotype.
• the one secreted by the GRB51D hybridoma has the IgG2a isotype.
• Titration of culture supernatants of cloned hybridomas by ELISA test made it possible to determine a culture supernatant of the hybridoma GRB98C of 1000. Those of the culture supernatants of the hybridomas GRB51D, GRA66D and GRA10D are 10 000.
• the title of the culture supernatant of the hybridoma GRA382E is 100,000. The high values of these titles should be noted.
• the protein (purified antigen: granzymes A or B, or negative control) is incubated in Laemmli buffer at 100 ° C for 10 min (1 ⁇ g / gel 12 cm wide), migrates on a denaturing polyacrylamide gel, then is electrotransferred on a nitrocellulose filter (BA83, CERA LAB0) in a graphite electrode device (CERA LABO), in a 39 mM glycine buffer, 48 mM Tris pH 8.3, 0.037% SDS, 20% methanol under a current of 1 mA / cm2 of filter for 1.5 to 2 hours. After transfer, the nitrocellulose filter is colored with 0.2% culvert red and 0.3% trichloroacetic acid to verify the effectiveness of the transfer, then cut into strips 0.6 cm wide.
• the strips are preincubated in a PBS buffer with 0.2% Tween 20 and 5% skimmed milk for 1 hour at 4 ° C.
• a set of strips, representing different proteins to be tested, are incubated overnight at 4 ° C. with the hybridoma supernatant diluted 1/10 in the PBS buffer containing 0.2% Tween 20 and 5% skimmed milk.
• the strips are then washed in PBS buffer containing 0.2% Tween 20 with moderate stirring for 5 times 5 min at laboratory temperature.
• the strips were incubated with an anti-mouse immunoglobulin coupled to alkaline phosphatase antibody (diluted 1/1000 in PBS buffer above) for 2 hours at laboratory temperature.
• the strips are equilibrated in the alkaline phosphatase reaction buffer (100 mM Tris pH 9.5, 100 mM NaCl, 50 mM MgCl 2 ) and placed in the presence of the phosphatase substrate (10 ml of reaction buffer, 44 ⁇ l of nitro-blue tetrazolium chloride, 33 ⁇ l of (5-bromo-4-chloro-3-indolylphosphatase p-toluidine) salt for 15 minutes at room temperature.
• the reaction can be stopped by washing the strips with H 2 O.
• Granzyme B reduced and denatured, is electrotransferred on a nitrocellulose filter.
• the nitrocellulose strips are incubated with the various antibodies to be tested, then with an anti-mouse antibody conjugated to alkaline phosphatase.
• the LGLs are placed in culture (medium: RPMI methionine depreciated, supplemented with methionine 35 s: 20 to 50 ⁇ Ci / ml and in 1% inactivated SHN) at a rate of 3 to 5 ⁇ 10 5 cells per ml.
• the labeling can be carried out in an RPMI medium which has been leucine-depleted and supplemented with 20 to 50 ⁇ Ci / ml of 3H leucine. After a 4 hour incubation at 37 ° C, the cells are washed twice in PBS.
• the cell pellet is taken up in 500 ⁇ l of RIPA-0.01% BSA (B2518, SIGMA), then sonicated for 15 s (micro sonicator, power 40 watts, probe 0.3-87, BIOBLOCK). The incorporation of radioactivity is followed by counting a fraction precipitated with TCA. The cell extract is aliquoted at 5 x 10 6 cpm.
• the eluate is incubated for 4 hours at 4 ° C. with shaking, in the presence of 200 ⁇ l of culture supernatant to be tested; 40 ⁇ l of protein A Sepharose R and 20 ⁇ l of RIPA-0.01% BSA (B2518, SIGMA).
• the antigen-antibodyprotein A Sepharose R complex is filtered, then washed, eluted with 45 ⁇ l of Laemmli buffer and boiled for 10 min at 100 ° C.
• the samples are deposited on a polyacrylamide gel (12.5%) in the presence of SDS. After migration to 40 mA 200 V, the gel is fixed, treated with EN3HANCE (DUPONT de NEMOURS) washed in distilled water, dried for 1 hour at 80 ° C and fluorographed on Hyperfilm-MP (AMERSHAM) at -80 ° C.
• EN3HANCE DUPONT de NEMOURS
• a band is observed at 31 KD corresponding to the migration of the LGL cell extract, immunoprecipitated by the anti-granzyme B antibodies, GRB51D and GRB98C. This band at 31 KD is also observed for the same cell extract immunoprecipitated by the anti-granzyme B polyclonal serum. On the other hand, this band is not detected with a negative control.
• the migration of the REX cell extract (cells not expressing granzymes) immunoprecipitated by the antibodies described above does not reveal any band at this molecular weight.
• JANSSEN tetra-methyl-pentadecane
• the protein A column stored in 0.02% Na azide, is washed with 5 ml of binding buffer. 2 ml of ascites, diluted 1/2 in the binding buffer, are placed on the column. After washing with 15 ml of binding buffer, the IgGs (immunoglobulins G) are eluted with 5 ml of elution buffer. This column is regenerated with 8 ml 0.1 M citric acid pH 3 and stored in 0.02% Na azide.
• the eluted fractions are desalted on Exocellulose columns with 10 ml of PBS. These desalting columns are regenerated with 15 ml of PBS and stored in 0.02% Na azide.
• the LGLs were incubated with antibodies recognizing granzymes A and B, selected by the ELISA test as reported above.
• the analysis is done by light microscopy.
• GVH graft-against-host reaction
• the tissue sections are saturated with horse serum (Vectastin kit, VECTOR) for 20 min at room temperature. After washing in PBS, 2.5 ⁇ g of the antibody to be tested are deposited on the section for 30 min at room temperature. The results of experiments carried out with the anti-granzyme antibodies GRA51D and GRA66D are reported.
• the slides are washed 3 times in PBS and then incubated with 50 ⁇ l of anti-Ig antibodies from biotinylated mice (Vectastin kit) for 30 min at room temperature, then with avidin mixed with biotinylated peroxidase (Vectastin kit) for 1 hour at room temperature.
• the revelation is carried out by incubating for 10 min at room temperature the slide covered with 50 ⁇ l of a solution containing the substrate (1 tablet (10 mg) of diaminobenzidine, dissolved in 10 ml of 50 mM Tris pH 7.6, mixed immediately with 100 ⁇ l of 0.03% H 2 O 2 ).
• the slides are washed and the tissue sections are stained with Meyer's Hemalun for 1 min at room temperature.
• the tissue sections are washed, dehydrated in ethanol baths (70%, 90% and 100%), passed through toluene and mounted with a drop of mounting resin. The analysis is done by light microscopy.
• a 1400 bp Smal-EcoRV fragment is excised from a complete mouse perforin cDNA clone. This segment codes for the C-terminal part of the perforin of mature mice covering residues 98 to 534 (see single figure).
• the blunt end of the segment is ligated into the BamHI site of the vector pAR3039 using phosphorylated linkers (5 '- CCG GAT CCGG-3').
• This plasmid is called pAR3039-perf.
• E.coli DE3 bacteria are transformed with this plasmid which contain in their genome the T7 polymerase gene under the control of an inducible promoter
• the recombinant 45 kDa perforin is purified by lysing the bacterial pellet (from 100 ml of culture) with 20 ml of a solution containing 50 mM Tris-HCl, pH
• the bacterial DNA is then fragmented by sonication and the protein inclusion bodies are centrifuged for 15 minutes at 12,000 rpm. The pellet is washed three times with a solution of 50 mM Tris-HCl, pH 7.5, 0.5 mM EDTA and 300 mM NaCl and dissolved in SDS-PAGE buffer.
• the 45 kDa recombinant protein is separated from the majority of bacterial proteins by SDS-PAGE (10% polyacrylamide gel) and subjected to electroelution using the ISC0 protein elution device.
• 1 to 2 mg of recombinant perforin is routinely obtained from 100 ml of bacterial culture.
• Maize OFA rats are immunized intraperitoneally with 50 ⁇ g of murine recombinant perforin (or rMup) and then by a second injection, intraperitoneally, 3 weeks later. 3 days before removing the splenocytes (for the production of hybridomas), 50 ⁇ g of rMuP are injected into the tail vein without adjuvant. 2) Production of hybridomas and selection:
• the fusion of Sp2 / O-Ag86 myeloma cells not producing immunoglobulins is carried out with splenocytes from rats immunized with rMuP. Briefly, the splenocytes are recovered by introducing carefully into the tissues of the spleen with a needle and a syringe of RPMI medium devoid of serum. The splenocytes are washed with the medium as carried out with the myeloma cells.
• 108 splenocytes are then mixed with 107 myeloma cells in a 50 ml FALCON tube and subjected to centrifugation for 10 minutes at 1000 rpm. We eliminate as much supernatant as possible.
• the cells are resuspended by gently tapping the walls of the tube and 0.4 ml of hot polyethylene glycol 1500 50% (w / v) (PEG, Serva, cat. No. 33123) is added dropwise while maintaining the tube in a water bath at 37 oC. After the addition of all the PEG, the tube is maintained at 37 ° C for 3 min and then centrifuged at 800 rpm for 5 min.
• PEG polyethylene glycol 1500 50% (w / v)
• RPMI medium devoid of serum (at 37 ° C.) is added over 2 min, then again 5 ml at once.
• the cells are centrifuged at 1000 rpm for 5 min.
• the supernatant is removed and 50 ml of complete RPMI medium - 5% FCS is added.
• the cell suspension is distributed into ten 96-well plates to which peritoneal monocytes / macrophages (feeder cells) from Balb / c mice (1 to 5 days before) have been added beforehand. The plates are incubated at 37 ° C.
• 96-well microtiter plates (Dynatech, MIC 2000) are covered for approximately 14 hours at 4 ° C. with 100 ⁇ l of rMuP (10 ⁇ g / ml in PBS).
• the wells are covered with granules derived from murine CTL B6.1 (solubilized in 1.5 M NaCl, ultracentrifuged and diluted 1/10 in water) or granules of human LAK cells (solubilized in phosphate 0.5 M, ultracentrifuged and diluted 1/5 in water). After washing the wells three times with 0.02% Tween-20 PBS, 100 ⁇ l of hybridoma supernatant is added to each well in 2 h at room temperature.
• the wells are washed as indicated above and 50 ⁇ l of goat anti-rat immunoglobulins conjugated with alkaline phosphatase (Sigma) are added in 1 hour. After washing, 100 ⁇ l of phosphatase substrate (p-nitrophenyl phosphate, Sigma 104 tablets) is added and the A405nm is measured as soon as the color begins to appear.
• phosphatase substrate p-nitrophenyl phosphate, Sigma 104 tablets
• the negative control wells are incubated with cRPMI-5% FCS instead of being incubated with supernatants of hybridomas. We thus recover the anti-perforin monoclonal antibody designated by the abbreviation CE2.10 deposited at the CNCM under the number 1-1058 on March 12, 1991.
• the fixed cells are resuspended in 1 ml 0.2% PBS.
• Tween 900 ⁇ l PBS + 100 ⁇ l of Tween 2% stock solution).
• washing solution 1 PBS 2% FCS, final 10 mM NaN3
• the suspension is centrifuged 8 min at 250 rpm.
• the supernatant is rejected; the cellular samples are then accessible for the study of intragranular proteins.
• an antibody buffer solution In order to reduce non-specific binding, 50 ⁇ l of an antibody buffer solution are added to the cell pellet.
• - Permeabilization control an anti-cytoskeleton antibody, the anti-tubulin ⁇ , (1a tubulin a being a strictly intra-cellular protein), is tested on all cell populations.
• washing solution (2) 0.2% PBS, Tween 20, 2% FCS, 10 mM final NaN3). Centrifugation is carried out 8 min at
• Tertiary antibody in order to carry out a simultaneous cytofluorometry study of the expression of perforin and granzyme B according to the phenotype and the functional state of the lymphocytes, an incubation with antibodies recognizing membrane antigens is carried out.
• anti CD3 at a rate of 0.125 ⁇ g (2.5 ⁇ l / test),. anti CD8 at 0.03 ⁇ g (2.5 ⁇ l / test) and
• the cells thus treated are resuspended in PBS-azide medium and analyzed within two hours. If the cells are analyzed later, they are returned to PBS-PFA 1% medium.
• This technique was used to compare the expression of granzyme B and perforin in umbilical cord blood lymphocytes with that of adult lymphocytes and assess cytoxicity in the case of transplants.
• the material used consists of frozen biopsies and bronchoalveolar washes for lung rejections.
• TBS buffer Solution A 100 ⁇ l + solution B 100 ⁇ l + 800 ⁇ l ED pH 7.6
• TEN 50 mM Tris and 0.5 mM EDTA + 300 mM NaCl DE3 strain (E.coli) containing a chromosome with the T7 polymerase gene under the control of a promoter inducible by IPTG
• the cultures are diluted to 1:10 in M9 + Amp. CAA, trp.
• the T7 RNA polymerase gene is induced with 0.4 mM IPTG.
• the cells are recovered and stored on ice before centrifugation carried out in 50 ml tubes, at 3500 rpm, 30 min at 4 ° C.
• the pellet is frozen to -20 ° C if necessary.
• the lysis is allowed to take place on ice for 30 min to 2 hours.
• the pellet is washed two to three times in TEA and then sonication is carried out to clarify the medium if necessary.
• the pellet is taken up in 500 ⁇ l of SDS buffer. For complete cell extraction, 1 ml of cell pellet is resuspended in approximately 200 ⁇ l of SDS buffer.
• a control is also prepared without induction by IPTG or a plasmid with an insert in a wrong orientation.
• nucleotide 116 is changed from A to T by mutagenesis in vitro, which results in a change in the amino acid site of granzyme 3 (from glu to val ).
• This construction allows the expression of the recombinant granzyme B whose sequence differs from that of the native human granzyme B in that it is preceded by Met and that amino acid 6 is not Glu but Val.
• the bacterial expression vector PAR 3038 uses the promoter of bacteriophage T7 to selectively express the recombinant proteins.
• the DE3 bacteria of the E. coli strain containing an endogenous chromosomal T7 polymerase gene under the control of a promoter inducible by IPTG, are transformed by the plasmid AR-3038 B.
• the cells are allowed to grow 2 to 3 hours until an absorbance at 600 nm of 0.8 to 0.9.
• the TA polymerase gene is then induced for 3 hours by the addition of 0.4 mM IPTG.
• the cells are collected and the recombinant protein is isolated from the insoluble protein fraction.
• the cell pellet (of 50 ⁇ l of culture) is resuspended in 5 ml of Tris Hcl (50 mM), 0.5 mM EDTA, pH 7.5, supplemented with 0.75 mg / ml of lysozyme and left on ice for two hours.
• the protein precipitate is recovered by centrifugation (6000 g, 30 min, 40 ° C) washed three times in 50 mM Tris-HCl, 0.5 mM EDTA, 300 mM NaCl, pH 7.5 and dissolved in SDS-PAGE buffer.
• the 25 kDa recombinant protein is separated from the main bacterial proteins by SDS-PAGE and electroeluted using the Biorad protein eluting device, following the manufacturer's instructions.
• Figure 4 gives the nucleotide sequence of human granzyme B and the corresponding amino acid sequence.
• a human perforin cDNA is used as isolated from a cDNA library (lambda ZAP-LAK) in a vector pBs / clone 15.
• the plasmid containing a cDNA insert of human perforin is called pAR 3039 HuP.
• This plasmid contains an ampicillin resistance gene.
• the construction method is shown in FIG. 5.
• the Sma / Eco RI cDNA insert comprises 1600 base pairs; this fragment codes for the amino acid sequence going from position Arg98 to Trp534.
• RNA Transcription into RNA is induced by IPTG.
• pAR 3039 is adapted with BamHI to Smal using as adapter GATCCCCGGG (Pharmacia).
• the Hu perforin Sma I fragment (from pBS / clone 15) is subjected to a ligation step.
• the open reading frame is controlled by sequencing.
• FIG. 6 represents the nucleotide pAR 3039 - HuP and the amino acid sequence deduced from the recombinant protein. The sequence of clone 34 is also indicated.

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