EP0575539A1 - Means for in-vitro diagnosis of constituents of cytoplasmic granules and biological applications - Google Patents

Abstract

The invention relates to means for the detection of constituents of cytoplasmic granules secreted by "helper" T cells or cytotoxic cells, in particular for the detection of granzyme or perforin, characterized in that they are monoclonal antibodies which recognize specifically an epitope of these constituents giving rise to an antigen-antibody type immunological reaction.

Description

 MEANS FOR THE IN VITRO DIAGNOSIS OF CONSTITUENTS OF

CYTOPLASMIC GRANULES AND BIOLOGICAL APPLICATIONS

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).

 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. 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.

In these granules, 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. During the interaction of the effector with its target and its activation, there is an exocytosis of the granular intracytoplasmic vesicles which secrete their content in the intercellular space formed by the effector / target contact, causing the lysis of the cell. target.

 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.

 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.

They contain all the residues His ⁵⁷ , Asp ¹⁰² and Ser195 which form the catalytic triad of serine esterases. All mature granzymes start with the N-terminal sequence Ile-Ile-Gly-Gly (residues 16-19), then a variable sequence (residues 20-23), followed by a conserved sequence (residues 24-30). These are more or less glycolysed proteins. Six conserved Cys residues form three intra-chain disulfide bridges.

 The molecular biology approach made it possible to isolate genes coding for granzymes expressed specifically in CTLs.

 Several roles have been assigned to granzymes.

In particular, an indirect action on perforin has been reported by increasing its lytic activity, promoting the fragmentation of the DNA of the target cell.

 They could also intervene in the mechanism of the detachment of the CTL from its target after the delivery of the lethal blow by cleavage of the TcR / antigen complex (TcR = T receptor to the antigen) and of the other receptor / ligand complexes involved in the phenomena of cell adhesion.

 Finally, 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.

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

Northern Blot and Dot Blot and more recently by the in situ hybridization technique using specific riboprobes.

 Studies carried out in vitro and, in vivo in a pathological situation in mice and in humans, show that these genes are inducible during the cellular activation of T lymphocytes, of cells exhibiting NK / LAK activity, and this early. In humans, the expression of the genes of granzymes and perforin has been observed in different pathological situations such as hematological disorders, autoimmune diseases, dermatological diseases and parasitic, bacterial or viral infectious diseases. Finally, the mRNAs of the granzymes and or of the perforin could be detected by in situ hybridization at the site of several types of allografts.

Expression of the granzyme B gene has been observed in cells infiltrating kidney allografts. The expression of the granzyme B and / or perforin genes could be detected in the cells preferably CD8 ⁺ in the lymphocyte infiltrates of endomyocardial biopsies carried out in patients with rejection. A similar study was carried out on the biopsies of the skin of patients with GVH (Graft versus host reaction) after genogenic or pheno-identical HLA allogeneic marrow transplantation.

 Studies have made it possible to observe when a rejection is suspected during an organ transplant

(heart, kidney) or GVHD after bone marrow transplant, the presence of "silent" 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.

 We therefore measure the value of the search for gene expression of granzymes and perforin during cellular and / or cytotoxic activation in vivo.

 Most of the studies carried out were carried out by Northern Blot, Dot Blot, in situ hybridization. Although informative, these techniques are cumbersome and do not allow in any case to study a large number of patients with the follow-up necessary to know the evolution of their pathology.

 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.

 It is clear that in all of these clinical applications, rather than detecting messengers of granzymes and perforin, it would be easier to detect proteins using antibodies.

 The fundamental aspect which involves understanding the biological role of granzymes and perforin also requires a fine and efficient analysis using antibodies.

 Taking advantage of their technical advance in this field and their great mastery of the techniques of isolation and purification of granzymes and perforin, the inventors have developed new tools making it possible to diagnose in humans the presence of constituents of cytoplasmic granules in biological samples to study.

Using the immunological approach to solve the problems posed to date, the inventors have produced high specificity antibodies against the above proteins.

 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.

 It also aims at a diagnostic method making it possible to simplify the detection of these products in clinical and fundamental research.

 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 expression "constituent of granules" as used in the description and the claims covers both the native form of the constituent and an active recombinant form as obtained by conventional techniques of genetic engineering on the basis of the sequence. amino acids of granzymes or perforin in humans, or amino acid sequences deduced from the nucleotide sequences of human genes encoding these proteins. Thus the granzymes or the perforin to which reference is made are either in their native form, or in a recombinant form, comprising at least the active part of these granzymes or perforin.

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.

 For the immunization step, 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 Krahenbϋhl et al. in J. Immunol, 141, 3471-77, 1988.

The constituents of the granules are in native form or, alternatively, in recombinant form. In particular, a recombinant human granzyme B or a recombinant human perforin is used. In the description and the claims, the expression "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 same is true for 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.

In accordance with the conventional technique, the hybridomas obtained are cultured and cloned according to the limiting dilution method. Advantageously, the hybridomas whose culture supernatants have the highest titers of antibodies in ELISA are selected. To increase the production of antibodies, they are injected into animals, for convenience, mice, rats or rabbits and ascites are thus produced in large quantities. As a variant, 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

D.S.M. (Deutsche Sammlung von Mikroorganismen) March 13, 1992.

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.

 The process for obtaining these hybridomas also falls within the scope of the invention. This method comprises the fusion and selection steps defined above in relation to the monoclonal antibodies.

 As indicated above, the 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.

 More specifically, these are amino acid sequences containing at least the active C-terminal part of the mature protein.

 Mention will in particular be made of the recombinant human granzymes B and the recombinant human perforin.

 By operating according to conventional techniques of genetic engineering, 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.

These 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.

 The implementation of these techniques leads to the development of tools which constitute new products. Thus, 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.

As indicated above, 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.

 In this application, the monoclonal antibodies are free. Alternatively, they are fixed on a non-immunogenic solid support.

 The method according to the invention for in vitro diagnosis of the presence of granzymes or of perforin or other constituent of said cytoplasmic granules is characterized in that it comprises the following steps:

 - 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.

To reveal the immunological reaction, said constituent, more particularly granzyme or perforin, comprises a marker group. It is most generally a radioactive group. Alternatively, 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.

 Thus it is particularly suitable for diagnosing a rejection in the case of organ transplantation (kidney, heart, / lung, liver, pancreas) and for the purposes of early diagnosis of GVH [skin, liver, intestine]) in the case bone marrow transplant.

 In addition to these immunohistological applications, 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.

 They could, for example, make it possible to follow the fate of a transplant in a patient, and in particular the implications of the immunosuppressive treatment which could possibly be modulated. This surveillance could be carried out by the kinetic study of "lymphocyte infiltrates" present at the level of biopsies taken at the graft site. They are also of great interest in the case of autoimmune, dermatological, infectious (microbial, parasitic or viral (HIV)) diseases.

 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.

 They can also be used to carry out cell differentiation studies, the granzymes being markers of T or B cell differentiation.

 It should be noted that in the case of autoimmune diseases the proteases involved constitute autoantigens.

They can therefore be used to recognize the autoantibodies formed. In this regard, 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. To carry out this reaction, 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.

 This kit is characterized in that it includes:

 - an appropriate solid phase serving as a support for the assay, such as a micro-titration plate,

 - a preparation of monoclonal antibody or fragment, free or immobilized, as defined above,

 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,

 - a specific detection system for the marker, appropriate buffer solutions for immunological reactions and for detection reactions.

 Monoclonal antibodies are also capable of constituting therapeutic agents of great interest and can play the role of vectors of enzymes or drugs.

 In the examples which follow, other characteristics and advantages of the invention are reported. Figures 1 to ... show respectively

 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,

- Figure 4, the nucleotide sequence of human granzyme B and the corresponding acid sequence amines (1 to 227) used to form recombinant human anti-granzyme B monoclonal antibodies,

 FIG. 5, the mode of construction of the plasmid pAR 3039-HuP, and

 - Figure 6, the nucleotide sequence pAR 3039

HuP and the deduced amino acid sequence of the recombinant protein, as well as the sequence obtained from clone 34.

 I - Hardware

1-1 Hardware

- Granzymes A and B are separated from the other proteins of LGL cytoplasmic granules (large granule lymphocytes) on a Mono S cation exchange column (HR / 5, PHARMACIA) connected to an FPLC system (PHARMACIA) according to the technique described by (Krëhenbϋl et al., In J. Immunol, indicated above).

 - The mice used are BALB / C mice of haplotype H-2d.

 - NS-1 myeloma, a non-secretor of the H-2d haplotype, brings fusion power to fusion. It is counter-selected in HAT medium (Hypoxanthine, Aminopterin, Thymidine) because it carries the HPRT- mutation (Kohler et al., Eur. J. Immunol. 6,292-295, 1976).

 - The polyclonal antibody directed against granzyme B is as obtained according to the technique described by Krâhenbϋhl (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),

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).

 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.

 Immunoprecipitation wash buffer: 100 mM

Tris pH 9, 0.5 M LiCl, 1% mercaptoethanol.

Laemmli buffer (Laemmli, Nature 227, 680-685, 1970): 62.5 mM Tris pH 6.8, 2% SDS, 15% glycerol, 5% β-mercaptoethanol, 0.01% bromophenol blue. II - Methods

II-1 Isolation of LGL from peripheral blood

The procedure is as described by Chouaib et al., In Proc. Natl. Acad. Sci. USA 85, 6875-6879, 1988.

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. After pouring 4 ml of the 41% solution and delicately depositing on it 2 ml of each solution by decreasing concentration in 15 ml conical tubes (NUNCLON DELTA, PAUL BLOCK), 3 x 10 ⁸ lymphocytes contained in 2 ml of RPMI are deposited on the surface of the gradient. The tubes are centrifuged for 30 min at 1400 rpm without brake at room temperature. The LGL rings located at the 37 - 41% interface are collected with a Pasteur pipette, washed 3 times in RPMI, then grouped. The cells are counted and cultured in medium 2 at the rate of 5 × 10 ⁵ cells per ml. LGLs represent 2 to 5% of the total lymphocyte population.

II-2 Immunization of mice with granzymes A and B purified from LGL from peripheral blood or from NK cells. 10 μg of purified proteins (granzyme A and B) dissolved in 200 μg 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 μg of protein dissolved in 200 μg of physiological saline).

II-3 Cell fusion We operate as described by Oi and Herzenberg, in

Selected methods in cellular immunology. Freeman and co. Mishell, Shiigi, eds. 1980)

Three days after the booster, 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. 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 μl per well (time 0).

 24 hours after the fusion (day 1), 100 μl of medium 5 (HAT selection) preheated to 37 "C is added to each well. This medium selects the hybridomas and kills the myeloma cells which have not fused.

 On day 7, 100 μl of medium are aspirated and replaced with 100 μl of new medium 4. On day 10, the content of the positive wells (ELISA test; AMERSHAM kit) is transferred to a flat-bottom plate of 24 wells in which 1 ml of culture medium is added. On day 13, the secretory hybridomas can be preserved by freezing in liquid nitrogen in SVF / 10% DMS0. II-4 Search for hybridomas secreting anti-human enzyme A and B antibodies by ELISA test (Enzyme Linked Immuno Sorbent Assay).

 (Mouse anti-IG kit, whole antibody linked to horseradish peroxidase, AMERSHAM)

The preparation of the 96-well flat-bottom plates is carried out 24 hours before the test. For this purpose, 100 μl of antigen (purified granzymes A or B), at 1 μg / ml (in

PBS) are distributed in the wells. The plates are placed at 4 ° C for 24 hours.

 2 to 5 washes with 200 μl per well of PBS, 0.1%

Tween 20 are performed before testing the culture supernatants of the hybridomas. The plates are incubated for 1 hour at

37 ° C with 50 μl per well of culture supernatant of the hybridoma to be tested.

 After 3 washes with PBS, 0.1% Tween 20, 50 μl of secondary antibody (conjugated with peroxidase and diluted 1/500 © in PBS, 0.2% Tween 20) are added to each well. Incubation is carried out at 37 ° C for 30 min.

 The plates are again washed 3 times with

PBS, 0.1% Tween 20. The development is carried out by adding 100 μl of a solution containing the substrate (10 μl 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.

 After fusion of the NSI myeloma cells with the splenocytes originating from the mouse immunized with granzyme B, by operating as indicated above, 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 same approach followed with granzyme A, led to the isolation of 15 hybridomas called GRA secreting antibodies specific for granzyme A, in an 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.

II-5 Cloning of hybridomas by limiting dilution.

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 μl of medium 4 per well.

 The cells are fed 3 days after cloning with 100 μl of medium 4, then every 7 days. When cell growth, controlled on an inverted microscope, seems significant, 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.

 Two 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.

II-6 Characterization of the isotype of cloned antibodies

The culture supernatant of hybridoma to be tested is diluted ^1/10 in 20 mM Tris pH 7.6, 137 mM NaCl (TBS). 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.

II-7 Determination of the specificity of these antibodies by the techniques of electro-transfer of proteins and of immunorevelation (Western blot). The procedure is as described by Towbin et al., In Proc. Natl. Acad. Sci.

76, 4350-4354, 1979.

The protein (purified antigen: granzymes A or B, or negative control) is incubated in Laemmli buffer at 100 ° C for 10 min (1 μg / 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. After 5 washes of 5 min in PBS, 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 μl of nitro-blue tetrazolium chloride, 33 μl 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.

 Under these conditions, a band at 31 KD is observed on the strip incubated with the anti-granzyme B polyclonal serum. On the other hand, this band is not detected on the strips incubated with the anti-granzyme B antibodies GRB51D and GRB98C. The controls (strips incubated with the antibody directed against the SV40 T antigen, with the anti-granzyme A antibodies GRA66D, GRA382E and GRA10D) are also negative. Anti-granzyme B antibodies therefore do not recognize granzyme B under the conditions of this Western blot experiment.

II-8 Labeling of cells in vitro and immunoprecipitation. The LGLs are placed in culture (medium: RPMI methionine depreciated, supplemented with methionine 35 s: 20 to 50 μCi / 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 μCi / 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 μl 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.

- Immunoprecipitation (according to Kress et al., In J. Virol. 31, 472-483, 1979).

Incubation, for 30 min at 4 ° C with shaking on a wheel, of the cell extract with 60 μl of protein A SepharoseR (PHARMACIA, CL4B SepharoseR) makes it possible to reduce the background noise. The sample is then filtered on a fry (porous polyethylene). 10 μl of normal mouse serum and 30 μl of protein A Sepharose ^R are added to the eluate and left for 1 hour at 4 ° C. with stirring. This solution is filtered on the same filter. The last step in reducing the background noise is carried out by adding 30 μl of protein A Sepharose ^R to the filtrate and an incubation for 30 min at 4 ° C. with shaking. After filtration, the eluate is incubated for 4 hours at 4 ° C. with shaking, in the presence of 200 μl of culture supernatant to be tested; 40 μl of protein A Sepharose R and 20 μl of RIPA-0.01% BSA (B2518, SIGMA). The antigen-antibodyprotein A Sepharose ^R complex is filtered, then washed, eluted with 45 μl 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.

The results of immunoprecipitation experiments followed by separation on acrylamide gel are reported below, carried out to verify the capacity of the monoclonal anti-B-enzyme enzymes to immunoprecipitate the B-enzymes contained in the LGL granules. In this experiment, cell extracts (LGL or REX) labeled with 35s-methionine are used. Immunoprecipitation is carried out using culture supernatants from hybridomas to be tested. The immunoprecipitated proteins are separated on a 12.5% denaturing polyacrylamide gel. The autoradiography time is 7 days.

 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. These results show that the GRB51D and GRB98C antibodies recognize granzyme B.

 For the characterization of anti-granzyme A antibodies (GRA66D, GRA382E, GRA10D), the immunoprecipitations were carried out on extracts of LGL and REX cell lines labeled with leucine 3H; indeed the amino acid sequence of granzyme A is poor in methionine, but on the contrary it contains 26 leucines. The immunoprecipitated proteins are separated on a 12.5% denaturing polyacrylamide gel. The autoradiography time is 1 month. An additional low intensity band at 32 Kd is observed only with the LGL cell extract immunoprecipitated by the antibody GRA66D, but not by the antibodies GRA382E and GRA10D, nor by an antibody serving as a negative control. II-9 Production of ascites

An injection of 500 μl of tetra-methyl-pentadecane (JANSSEN) is carried out intraperitoneally to a BALB / C mouse. The procedure is as described by Hoogenraad and Wraight, in Methods Enzymol, 121, 375-381, 1986. Ten days later, 107 hybridomas washed in physiological saline are injected intraperitoneally. The mice are punctured every day. Peritoneal fluid is centrifuged for 7 min at 1500 rpm at 4 ° C. The ascites recovered using a Pasteur pipette is filtered through 0.2 μm (NALGENE), then centrifuged at 4 ° C for 30 min at 12000 g to remove the lipids and stored at -80ºC.

II-10 Purification of antibodies

 (ImmunoPure IgG Purification kit, PIERCE)

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 desalted fractions of IgG are assayed by measuring the OD at 280 nm to determine the concentration of immunoglobulins in each fraction according to the formula: [IgG] in mg / 1 = OD at 280 nm / 1.4. II-11 Detection of granzymes at the LGL level by immunocytochemistry.

The LGLs were incubated with antibodies recognizing granzymes A and B, selected by the ELISA test as reported above.

2 x 10 ⁵ cells per slide are cytocentrifuged for 5 min at 250 rpm and fixed in acetone for 10 min at 4 ° C. After 2 washes in PBS, 10 μl per slide of the antibody to be tested (diluted 1/2 in PBS, 1% BSA) are added and left for 1 hour at 37 ° C. The slides are washed 2 times in PBS and then 200 .mu.l per blade of the anti mouse Ig antibody (diluted ^1/500 in PBS, 1% BSA) are added and left 30 min at 37 ° C. After 2 washes in PBS, the revelation is carried out by incubating for 10 min at room temperature the slide covered with 200 μl of a solution containing the substrate, 1 tablet (10 mg) of 3.3 ′ diaminobenzidine (SIGMA) dissolved in 10 ml of 50 mM Tris pH 7.6, mixed immediately with 100 μl of 0.3% H2O2. The slides are washed and the cells are stained with Meyer's Hemalun (MERCK) for 1 min at room temperature. The cells are washed, dehydrated in ethanol baths (70%, 90% and 100%), passed through toluene and mounted with a drop of mounting resin (BIOLYON).

 The analysis is done by light microscopy.

 Under these conditions, a brown coloration of the cytoplasm is observed, revealing the binding of these antibodies. No staining is obtained with an antibody used as a negative control. When LGLs are activated overnight in the presence of recombinant IL-2 (250 U / ml), only part of the cell population is stained. After seven days of culture in the presence of IL2 at the same concentration, the entire cell population is colored.

II-12 Detection of granzymes A and B on skin biopsies of patients with GVH by immunohistochemistry.

During an allogeneic marrow transplant performed in HLA identity, the clinical syndrome of the graft-against-host reaction or GVH is frequently observed. The clinical manifestations of GVH are characterized by damage to the skin, the intestine and the liver. Skin biopsies are considered to be the best material for establishing the diagnosis of GVHD through histology and immunohistochemistry. The biopsies of 2 patients (RAP: chronic myeloid leukemia (CML); REN: acute myeloid leukemia (AML)) presenting lesions compatible with GVH were studied. Skin biopsies frozen in OCT (Tissue-Tek, MILES), fixed in acetone for 10 min at 4 ° C, are cold cut to 5 μm. The tissue sections are saturated with horse serum (Vectastin kit, VECTOR) for 20 min at room temperature. After washing in PBS, 2.5 μg 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 μl 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. After 2 washes in PBS, the revelation is carried out by incubating for 10 min at room temperature the slide covered with 50 μl 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 μl 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.

 As previously observed in vitro, in the case of LGL, a staining of the cytoplasm of lymphocytes infiltrating the skin. No staining is obtained with the control antibody used as a negative control.

In addition, the coloration indicating the fixation of the various anti-granzyme antibodies is found to be localized at the level of the lymphocyte infiltrates which have been characterized in immunohistochemistry by the membrane markers of lymphocyte differentiation (CD3, CD4, CD8). III - Production of recombinant mouse perforin. To produce recombinant mouse perforin, a prokaryotic expression vector pAR3039 is used which expresses the proteins under the control of the transcription and translation signals of phage T 7 (see Studier et al.,: J. Mol. Biol. , 189, 113-130, 1986).

 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

IPTG. Corresponding bacteria have been deposited at the

C.N.C.M. March 12, 1991 under No. 1-1057.

 The synthesis of recombinant perforin is induced by IPTG in the transformed bacteria.

 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

7.5, 0.5 mM EDTA and 10 μg / ml lypozyme for 12 hours on ice.

 After adding 1.5 ml of 5M NaCl and 1.5 ml of 10% NP-40, the lysed bacteria are kept for 20 minutes on ice.

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.

IV - Production of the anti-perforin monoclonal antibody CE2.10 1) Imimization process:

Maize OFA rats are immunized intraperitoneally with 50 μg 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 μg of rMuP are injected into the tail vein without adjuvant. 2) Production of hybridomas and selection:

By operating as described by Harlow and Lane in Cold Spring Harbor Laboratory - New York, 1988, 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. Without removing the supernatant, 5 ml of 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. in a humidified atmosphere at 5% CO 2 for 24 hours, before replacing the medium with a fresh medium cRPMI-5% FCS supplemented with 1% HAT (Gibco). The cells are allowed to grow for about 10 days. At this stage, about 70% of the wells have clones that have grown, many of them having more than one clone. About 20 days after fusion, the cells in the majority of the wells containing hybridomas have reached a practically maximum density and 100 μl of each well are removed to carry out an ELISA test on rMuP and native murine perforin. Strong positive hybridomas are subcloned as described in the Harlow article mentioned above and subjected to another selection operation.

Selection by ELISA test to obtain a monoclonal anti-perforin antibody.

96-well microtiter plates (Dynatech, MIC 2000) are covered for approximately 14 hours at 4 ° C. with 100 μl of rMuP (10 μg / ml in PBS). In alternatively, 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 μl of hybridoma supernatant is added to each well in 2 h at room temperature. The wells are washed as indicated above and 50 μl of goat anti-rat immunoglobulins conjugated with alkaline phosphatase (Sigma) are added in 1 hour. After washing, 100 μl of phosphatase substrate (p-nitrophenyl phosphate, Sigma 104 tablets) is added and the A405nm is measured as soon as the color begins to appear. 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.

V - Study of the expression of perforin and granzyme B in flow cytometry.

The results obtained by following the slightly modified method of Schmid I et al. Are reported below. Cytometry, 12: 279-285, 1991.

Material: All the solutions are prepared in PBS.

- Paraformaldehyde 3% stock solution (PFA),

 - Fetal calf serum (SVF) filtered at 0.2 μ,

 - Tween 20. 2% stock solution,

- Arid NaN3. Stock solution at 200 mM Technical:

Fixation :

 1.106 cells washed in PBS are resuspended in 850 μl of PBS (4 ° C), 150 μl of PFA (4ºC) are added and the suspension homogenized. The samples are incubated at 4ºC for 1 hour, then centrifuged 8 min at 250 g.

Permeabilization:

 The fixed cells are resuspended in 1 ml 0.2% PBS. Tween (900 μl PBS + 100 μl of Tween 2% stock solution).

 The samples are then incubated for 15 min at 37 ° C. 2 ml of washing solution 1 are then added (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.

Incubation:

 primary antibody:

 In order to reduce non-specific binding, 50 μl of an antibody buffer solution are added to the cell pellet.

 It is resuspended, then 50 μl of 1/3000 antiperforin antibody (ie 1/6000 final) and of 0.25 μg anti-granzyme B antibody are added.

Cookies used:

 negative control: an irreversible mouse immunoglobulin, of the IgG1 type is used on the cell suspension to be studied.

- Permeabilization control: an anti-cytoskeleton antibody, the anti-tubulin α, (1a tubulin a being a strictly intra-cellular protein), is tested on all cell populations.

 - Cell positive control: cultured LGL cells,

 - cell negative control: lymphoid line

MOLT 4.

 The samples thus treated are incubated for 30 min at 4ºC, then washed twice in washing solution (2)

(washing solution (2) = 0.2% PBS, Tween 20, 2% FCS, 10 mM final NaN3). Centrifugation is carried out 8 min at

250 g.

Secondary antibody: 60 ng of GAM are added in 50 μl of PBS to the cell pellet resuspended in 50 μl of antibody buffer. It is left to incubate for 30 min at 4 ° C., then two solution washes are carried out (2).

 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.

 We use antibodies

 . anti CD3, at a rate of 0.125 μg (2.5 μl / test),. anti CD8 at 0.03 μg (2.5 μl / test) and

. anti CD25 at 7.5 μl / test

in a volume of 50 μl PBS.

 50 μl of antibody buffer and 50 μl of antibody are added to the cells. The mixture is incubated for 30 min at 4 ° C., then two washes are carried out using a PBS-azide mixture.

 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.

VI - Clinical applications:

 The results of studies concerning the use of the antibodies of the invention in various pathological situations such as:

 - heart rejections after transplantation,

 - rejection of lungs after transplantation.

 These two models make it possible to study the cytotoxic activation of cells which infiltrate a graft.

 This approach allows it to be extended to all models of transplant rejection whatever they are. The material used consists of frozen biopsies and bronchoalveolar washes for lung rejections.

 Other models involving a role of cytotoxic cells in the pathology itself or in the immune response generated by this pathology have also been studied, namely

 - skin tumors,

 the T cell response in non-Hodgkin's lymphoma.

 The technique used in all these models is the APAAP technique, in three layers

We follow the following protocol:

 1 ° - thawing of the slides kept in the freezer at room temperature 15 min,

 2 ° - writing on each slide in pencil of the antibody to be tested,

 3 ° - fixation in an acetone bath 10 min at room temperature,

 4 ° - drying 3 - 4 min,

 5 ° - rehydration 10 min in TBS pH buffer

7.6, 30% of SVF,

6 ° - application of a first layer and incubation at room temperature in a humid chamber for 30 min, 7º - rinses in TBS pH 7.6 twice 5 min,

 8 ° - incubation with the second layer (rabbit anti-mouse Ig antibody),

 9th - rinses two to three times in TBS pH 7.6,

10 ° - application of a third layer of antibody (P.A. complex coupled to an anti P.A. mouse antibody 30 min in a humid chamber),

 11 ° - double rinses in T.B.S. pH 7.6 then T.B.S .. pH 8.2,

 12 ° - revealing of alkaline phosphatase 20 to 30 min in the dark,

 13 ° - rinsing,

 14 ° - counter-coloring,

 15 ° - mounting in an aqueous medium.

Products used:

 * T.B.S. 0.05 M, pH 7.6

solution A TRIS = 60.55 g in 11 H ₂ O distilled, pH 7.6

solution B + Nacl - 87.66 in 11 H ₂ O distilled, pH 7.6

* 0.05 M TBS buffer = Solution A 100 μl + solution B 100 μl + 800 μl ED pH 7.6

* Developer: for 100 ml of Tris

 1 ° / Naphtol ASTR phosphate 20 mg

 2 ° / Dimethylformamide 2 ml

 3 ° / TRIS pH 8.2, 0.01 M 100 ml

 4 ° / Levamisol 2.4 g for 10 ml of ED 130 μl

5 ° / Fast Red TR knows 100 mg

 67 Filtration * Monoclonal antibodies

second layer: Dako 10g 20%, Z 259 80% diluted in TBS

7.6 to use 1/20 of Ac2 20% SHN Ab

third layer: A.PAAP D 651 1/50 diluted in 20% SHN VII - Expression of the recombinant human granzyme B and of the recombinant human perforin in bacterial haste

(T7 PQL).

The procedure is as described above by Studier et al. in J. Mol. Biol (1986), 189, P 113-130.

MATERIAL Plasmid Upstream site Fusion Framework Termination

 downstream open site

 cloning pAR3038 Bal I BamHI (8 mothers) ATC To EcoRV pAR3039 Bal I BamHI (10 mothers) GAT To EcoRV

1 2 3 4 5 6 7 8 9 10 11

 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg

 CAT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGC ATC 'C Smères

CGG GAT C 'C 10mères Nd e ^I. NheI M9 + CAA, Trp. Middle amp

 M9 medium plus 2 g / l casamino acids

 3.3 ml tryptophan to

 10 mg / ml 50 mM Tris-Ci (pH 7.5) Stock: 1 M pH7.5

 0.5 m EDTA Stock: 0.5 M

 10 mg / ml freshly prepared lysozyme

 IPTG in sterile H2O (stock at 20 ° C, 20 mg / ml = 0, Q84M, diluted 210 times)

5 M NaCl

10% NP-40 in H ₂ O

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

 (1) Growth of cells:

 Example: E.coli DE3; T7 HLP; for analytical purposes the volume is reduced by 5 times.

 1. We start over approximately 14 h (inoculum) from freshly prepared plates (less than 48 h) of LB-Amp (50 μg / ml) in M9 + CAA, Trp. Amp. The sample is taken to deposit in a 100 ml Erlenmeyer flask. The volume must be 10% of the volume desired for the culture (it is also possible to grow the cells in LB).

 2. The cultures are diluted to 1:10 in M9 + Amp. CAA, trp. The growth is allowed to take place until a measurement of OD600 = 0.8-0.9 (usually 2 to 3 hours) at 37 "C with good aeration.

 3. The T7 RNA polymerase gene is induced with 0.4 mM IPTG.

 4. Let growth take place for 3 hours.

 5. The cells are recovered and stored on ice before centrifugation carried out in 50 ml tubes, at 3500 rpm, 30 min at 4 ° C.

 6. The pellet is frozen to -20 ° C if necessary.

 (2) cell lysis and preparation of insoluble protein fractions.

1. For 50 μl of culture, resuspend (50 mM Tris + HCl (pH 7.5), 0.5 mM EDTA, and add 150 μl 25 mg / ml of lysozyme (freshly prepared in water) .

 2. The lysis is allowed to take place on ice for 30 min to 2 hours.

3. 0.75 ml of 5M NaCl is added, which must lead to the lysis of the cells. A Vortex is used and then 0.75 ml 10% NP-40 is added. We re-use the Vortex. The mixture is very viscous and stored on ice for 10 to 30 min to obtain the lysis of all the cells. 4. A sonication treatment is applied to break the DNA (pulses of 2 x 10 - 15 sec. Until a liquid medium is obtained).

 5. We rotate the ppr proteins.

 6. The pellet is washed two to three times in TEA and then sonication is carried out to clarify the medium if necessary.

 7. The pellet is taken up in 500 μl of SDS buffer. For complete cell extraction, 1 ml of cell pellet is resuspended in approximately 200 μl of SDS buffer.

 A control is also prepared without induction by IPTG or a plasmid with an insert in a wrong orientation.

VIII - Recombinant human Granzyme B

 . Construction of the vector pAR - 3038 - GraB

All the cloning techniques used are carried out by operating according to Maniatis et al. in Molecular Cloning. A laboratory manual New York; Cold Spring Harbor, Laboratory Press - Schmidt et al., 1987 / J. Immunol. 139, 250-256.

 From an HLP clone, a complete cDNA insert of human granzyme B is isolated (mobilization of the insert with BamHI). The entire cDNA is subcloned into the BamHI site of ml3mp8.

 Using the Kunkel technique (Bio-Rad kit,

Kunkel et al. Methods in Enzymology, 1987, 154, 367, 382), 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 change in nucleotide generates a new Bail restriction site which makes it possible to isolate the Bail - BamHI fragment of the granzyme B clone corresponding to the amino acids of region 6 to 227, plus a region of untranslated nucleotides (see FIGS. 2 and 3). By ligation the Bail-BamHI fragment is introduced into the vector PAR-3038, which has been cut with Nde I and BamHI and dephosphorylated. We use a synthetic Ndel - Ball linker which includes the first 6 residues of granzyme B to adapt the Ndel site of the vector and the Bail site of granzyme B.

 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.

. Expression of the recombinant granzyme B

 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 synthesis of the recombinant protein is induced in DE3 cells, transfected with the plasmid PAR-3038. GraB as follows:

 an inoculum of approximately 14 hours, coming from plates to which L Broth has recently been applied - 50 μg / ml of ampicillin, is diluted to 1/10 in the same medium. 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.

 Briefly, the cell pellet (of 50 μl 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.

750 μl of 5 M NaCl and 750 μl of 10% BP-40 are added with vigorous stirring. The medium is maintained for 30 min on ice, then the bacterial DNA chains are broken by sonication (three pulses of 15 sec) until the solution loses its viscosity and becomes liquid.

 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.

 From 400 ml of bacterial culture, approximately 1 to 2 mg of recombinant protein is obtained repeatedly.

 Figure 4 gives the nucleotide sequence of human granzyme B and the corresponding amino acid sequence. IX - Recombinant human Perforin B:

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.

Transcription into RNA is induced by IPTG. For construction, 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.

Claims

1 / Antibodies directed against the constituents of the cytoplasmic granules of activated T "helper" cells or of cytotoxic cells, more especially cytotoxic T lymphocytes and "natural killer" cells, characterized in that they are capable of monoclonal antibodies to react specifically with an epitope of a given constituent of a human granule, in native form or in recombinant form, in particular an epitope of human granzyme or of human perforin, in native or recombinant form, giving rise to a compound of antigen-antibody type.

 2 / Antibodies according to claim 1, characterized in that the monoclonal anti-granzyme antibodies are monoclonal anti-granzyme A, B or H human antibodies.

 3 / Antibodies according to claim 1 or 2, characterized in that they belong to the class of IgG, that they are directed against proteins of molecular weight of approximately 25 to 30 kDa (granzyme B), 60 kDa

(granzyme A) or approximately 66-75 kDa (perforin).

 4 / Antibodies according to one of claims 1 to 3, as induced by a process comprising the steps:

 - fusion of non-secreting myeloma cells with cells producing antibodies directed against a given constituent of cytoplasmic granules of cytotoxic cells or of "helper" T cells, in particular of antibodies directed against granzymes or perforin.

 - selection of hybrid cells capable of producing specific antibodies with respect to said constituents,

 - cloning of these hybridomas,

and

- Purification of monoclonal antibodies as produced for example from ascites liquids or culture media. 5 / Antibodies according to claim 4, characterized in that the antibodies produced by the cells used in the fusion step are induced by constituents of granule in native form.

 6 / Antibodies according to claim 4, characterized in that the antibodies produced by the cells used in the fusion step are induced by constituents of granule in recombinant form, in particular by a recombinant human granzyme B or a recombinant human perforin.

 7 / Monoclonal antibodies characterized in that they are anti-human granzyme B and human anti-granzyme A monoclonal antibodies as secreted by the clones GRB 51D and GRA 66D deposited at the CNCM under the numbers I-1060 and I-1059 on March 12, 1991, or also of recombinant human anti-granzyme B antibody or recombinant human anti-perforin as secreted by the clones deposited at the DSM on March 13, 1992.

 8 / Hybridoma strains characterized in that they are capable of secreting monoclonal antibodies according to any one of the preceding claims.

 9 / hybridoma strains according to claim 8, characterized in that they are formed from hybrid cells resulting from the fusion of myeloma cells with cells producing specific antibodies after immunization with a given constituent of the cytoplasmic granules of cytotoxic cells, more especially with purified granzymes or perforin, or recombinant granzymes and perforin.

 10 / Clones producing anti-granzyme monoclonal antibodies deposited at the CNCM under the numbers 1-1060 and I-1059 on March 12, 1991 and at the DSM on March 13, 1992.

 11 / Clone producer of recombinant human anti-perforin monoclonal antibodies deposited with the DSM on March 13, 1992.

12 / Process for the preparation of a hybridoma according to any one of claims 8 to 11, characterized in that it comprises the merging and selection steps defined in claim 4.

 13 / A method according to claim 12, characterized in that the myeloma cells are NS1 myelone cells and that the cells producing monoclonal antibodies are splenocytes.

 14 / Method for in vitro detection of the presence of constituents of cytoplasmic granules of "helper" T cells or of cytotoxic cells, in particular of granzymes or of perforin in a biological sample, characterized in that it comprises:

 - bringing the sample from a patient likely to be suffering from a disease accompanied by the exocytosis of said constituents, in particular granzyme or perforin, into contact with a preparation of monoclonal antibody or a fragment of the latter, as defined above, under conditions suitable for the production of an antigen-antibody complex, and the detection of the immunological binding.

 15 / Kit for the in vitro detection of the presence of the constituents of the cytoplasmic granules of "helper" T cells or of cytotoxic cells in particular of granzyme or perforin in a biological sample, characterized in that it comprises:

 - an appropriate solid phase serving as a support for the assay, such as a micro-titration plate,

 a preparation of specific monoclonal antibody or fragment, free or immobilized, as defined above,

 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 such a group,

- a specific detection system for the marker, appropriate buffer solutions for immunological reactions and for detection reactions. 16 / Application of the monoclonal antibodies according to any one of claims 1 to 7 to the detection of the presence of constituents of the cytoplasmic granules secreted by cytotoxic cells.

 17 / Proteins of the cytoplasmic granules of "helper" T cells or of cytotoxic cells, characterized in that they are recombinant proteins as obtained, by genetic engineering, in cellular hosts, in particular bacteria, transformed by introduction expression vectors, in particular plasmids, containing gene fragments coding for the desired amino acid sequences.

 18 / DNA fragments characterized in that they consist of the coding sequence for a protein according to claim 17, or that they contain such a sequence, and that they are optionally incorporated in an expression vector such as a plasmid.

 19 / Expression vectors in particular plasmid containing a DNA fragment according to claim 18, and cellular hosts transformed by these vectors.