FR2686087A1 - NOVEL LYMPHOCYTATIC ANTIGEN, CORRESPONDING ANTIBODIES AND THEIR APPLICATIONS. - Google Patents

Abstract

Antigenic protein consisting of a 150 +/- 10 kDa glycoprotein determined by electrophoresis of the SDS-PAGE type in reducing conditions, expressed on the surface of T-cell lymphocytes in human blood. The present invention also concerns the protein in its dimeric form having a disulphide bond and a molecular weight of 300 +/- 20 kDa. The invention further relates to antibodies directed against a protein of the invention, hybridoma lineages and the diagnostic and therapeutic application of proteins and antibodies according to the invention.

Description

 The present invention relates to a new lymphocyte antigen as well as antibodies corresponding to this antigen and their applications.

Lymphocyte activation induces or increases the expression of several surface structures, some of which, such as the IL2 and transferrin receptors, are directly involved in cell growth. In particular, the activation of human lymphocytes leads to phenotypic changes appearing either immediately or at variable times after the disruption of the antigen recognition structures. Phenotypic changes can be attributed to expression on the cell surface inducible molecules such as CD25 (1,2) and CD71 (3), or to increased expression of surface antigens such as CD26 (4,5), CD 29 (6,7), CD45RO (8,9 ), or to an epitopic modulation as in the case of CD2 (10,11), or to changes in affinity as reported for the molecules CD11a / CD18 (12) and LAM1 (13). It is not surprising that these inducible molecules include receptor subunits of high affinity for various cytokines (2). Most often, they are detected using a monoclonal antibody (mAb) recognizing a putative receptor for an unknown ligand. For example, the ligands of CD5 (14,15), CD43 (16) and CD69 (17,18) have been very recently identified, when the use of mAb as agonists against these structures had made it possible to identify their crucial role in the activation of the T cell (15, 17, 18, 19). These mAbs have been shown to increase the cytoplasmic concentration of free calcium, if they are "cross linked" to the surface of T cells (19). Since an increase in cytosolic calcium and simultaneous activation of Protein Kinase C (PKC) are early and essential events in the proliferation of T lymphocytes, investigations have been carried out into the effects of these various
MAb on the activation of human T lymphocytes in the presence of PMA (Phorbol 12-myristate 13-acetate), which is an activator of PKC (20).

 In addition, the use of mAb has confirmed that a large number of leukocyte surface antigens and in particular those induced during activation are common to the B and T cell lines (2,3,14).

 In order to identify new structures characteristic of activated lymphocytes, monoclonal antibodies have been defined according to the invention against functionally defined clones of human T cells.

According to the present invention, a monoclonal antibody called BB18 was first isolated, recognizing, on the cell surface, a new lymphocyte antigen composed of 150 * 10 kDa subunits, the expression of which on T lymphocytes increases rapidly after their activation by various stimuli including lectins. On the other hand, in the presence of phorbol 12-myristate 13-acetate (PMA), the expression on the cell surface of this structure of 150 +10 kDa undergoes a negative modulation even earlier than the molecules of the
CD3. Biochemical studies as well as phenotypic analysis have revealed that this structure is different from all the molecules previously identified on the cell surface of lymphocytes.

 A second BD16 monoclonal antibody obtained according to the invention recognizes the same surface structure as the AMC BB18. These two antibodies are lgGl which were obtained after immunization of Balb / c mice with a human thymic clone and fusion with the cell line NS1. They immunoprecipitate from the surface of human peripheral blood lymphocytes or radio-labeled iodine T clones, the same molecular structure. The modulation of the surface structure by incubation with one or the other antibody induces the loss of fluorescence reactivity with the two antibodies. However, these two antibodies react with epitopes different from this structure because there is no d inhibition of the binding of the antibody BB18 in the presence of an excess of the antibody BD16 and vice versa.

 The present invention therefore firstly relates to an antigenic protein consisting of a glycoprotein with a molecular weight of 150 + 10 kDa determined by SDS PAGE type electrophoresis under reducing conditions, protein expressed on the surface of preferably activated human T lymphocytes .

 The present invention also relates to an antigenic protein consisting of the same glycoprotein but in the form of a disulfide-linked dimer of molecular weight of 300 + 20kDa determined by electrophoresis of SDS PAGE type under non-reducing conditions, protein expressed at the surface. preferably activated human T lymphocytes, composed of 150 + 1 OkDa subunits as defined above.

 The subject of the invention is also derivatives of the protein in monomeric or dimeric form, characterized in that it is in non-glycosylated form or in that it comprises non-natural glycosilations or a fragment of this glycosilated protein or not comprising essential protein antigenic sites.

 The present invention also relates to monoclonal antibodies recognizing an epitope of a protein according to the invention, in particular the monoclonal antibodies BD16 and BB18, as well as hybridoma cell lines producing these antibodies, in particular the lines deposited at the ECACC under the numbers 92010801 for the antibody BD16 and 92010802 for the antibody BB18.

 The proteins according to the invention can be obtained not only by purification from producer cells by immunopreclpltatlon techniques, in particular with antibodies BB18 and BD16 or other techniques, but also by recombinant DNA techniques from eukaryotic or prokaryotic cells, in which case the glycosilation of the recombinant protein obtained may differ from the natural protein as is known to those skilled in the art.

 A protein or an antibody according to the invention can be labeled with a detectable label with a view to their assay in vitro or their localization in vivo or ex-vivo.

 The invention indeed also relates to the use of proteins and antibodies according to the invention for the diagnosis of pathological conditions or the monitoring of the treatment of ailment or as a warning light for the appearance of these ailments. In particular, the present invention relates to the use of these proteins and antibodies for the rapid diagnosis of an early activation state of lymphocytal T cells.

Finally, the present invention relates to the use of a protein, antibody or antibody fragment according to the invention as a medicament in particular for the treatment of diseases with signs of deficit
Immune or inflammatory or autoimmune pathology and in the in vivo or ex vivo treatment of malignant lymphoid proliferation.

 Other advantages and characteristics of the present invention will become apparent in the light of the description which follows.

In the appended figures, - Figure 1 represents the kinetics of expression on the cell surface of
the structure recognized by BB18 after activation of the CMSP (cells
peripheral blood mononuclear) using PHA. Cells
peripheral blood mononuclear cells were marked before (OD), 3
days (D3), or 9 days (D9) after coculture with PHA I ug / ml,
for indirect immunofluorescence with BB18, CD25 "BC96" or a
lgGl negative monoclonal antibody. The fluorescence profiles are
figured as histograms.

- Figure 2 represents the PMA inducing an early decrease in
reactivity with BB18. Peripheral blood T cells have been
incubated with PMA I ug / ml for 6, 10, 24 or 144 h. Cells
have been washed and treated for indirect immunofluorescence with
various monoclonal antibodies. At each measurement time, a check
negative made of Incubated cells under the same conditions but without
PMA has been completed. As no changes in the
control cells, the control tested after 6 h of culture is presented. The
fluorescence profiles are shown as histograms.

- Figure 3 shows the SDS-PAGE profile of a BB18 immunoprecipitate
obtained from a lysate of cloned T cells. The immunizing clone B12
of human thymus T cells was labeled on the surface and lysed in
NP40 1%. The immunoprecipitates obtained with Bol8, CD18 "M232" or a
irrelevant lgGî monoclonal antibodies were analyzed on a gel
7.5% SDS-PAGE under non-reducing conditions. The positions of
molecular weight markers have been indicated on the left in kDa.

- Figure 4 represents the two-dimensional gel analysis (not
reducer / reducer) of an immunoprecipitate BB18 obtained from lysates
cloned T cells. Immunoprecipitates obtained with the antibody
monoclonal BB18 were separated on the edge under non-
reducing agents (N. Red.) on a 7.5 0 gel using a PHAST system.

Then the gel strip containing the immunoprecipitate was cut and
subjected to electrophoresis in the second dimension, as recom
requested by the manufacturer, after reduction (Reduction) on an SDS-PAGE gel at
7.5%. The positions of the molecular weight markers have been indicated
right in kDa.

- Figure 5 shows the kinetics of the proliferation of PBMCs induced
by the monoclonal antibody BB18. Blood mononuclear cells
device were incubated with purified CD3 "X3" 5 pg / ml (triangles
white), the purified monoclonal antibody BB18 5 μg / ml (empty squares) or
10 ugiml (white circles) in the presence of PMA 0.2 ng / ml for 2 to S
days. The incorporation of (3H) TdR was measured by "pulsing" the culture
during the last 16 hours. The results are expressed as the
average of triplicated samples and the SEM has always been less than
10oxo of the average. Under these conditions, the PMA alone did not induce
significant proliferation at any of the measurement times.

 In an attempt to define activation-specific cell surface glycoproteins, we have, according to the invention, used mAbs obtained by repeated immunizations with highly functional human T cell clones (21). In particular, an mAb was initially identified on the clone called B12 (22) of human thymus T cells.

called BB18, recognizing a new disulfide-linked dimer composed of subunits of 150 + 10kDa. This structure, which is weakly expressed in normal peripheral blood lymphocytes, quickly becomes strongly expressed during activation.

In contrast, incubation of CMSP with PMA induces rapid negative modulation followed by complete re-expression within 24 hours.

This molecule is not specific for the T cell because it is also expressed in EBV-transformed B cell lines. Moreover, the
CMSP proliferate when this dimer has been stimulated in the presence of submitogenic concentrations of P \ 'A.

 1. MATERIALS AND METHODS 1. Monoclonal antibodies.

The monoclonal antibodies used such as CD1 "L404", CD3 "OKT3", CD4 "0516", CDS "L533", CD8 "OKT8", CD18 "M232" and
CD25 "BC96" were either locally produced (23) or purchased commercially. The monoclonal antibody BB18 was prepared by Immunization of Balb / c mice with the thymic clone B12, CD4 CD8 (22) (three intravenous injections with 20 × 10 6 cells). Spleen cells from immunized mice were fused to the NUS 1 cell line five days after the last injection. Initial screening by indirect immunofluorescence and flow cytometry using a Facstar (Becton Dickinson, Mountain
View, CA) retained all the supernatants of hybridomas reacting with the immunizing cells but not or weakly with the mononuclear cells of the peripheral blood at rest. The culture containing the above-mentioned monoclonal antibody was cloned twice by limiting dilution. Serial passages of the cloned hybridoma were performed by lp injection in Balb / c mice sensitized with pristane. The ascites fluid was collected, ultracentrifuged and affinity purified, if necessary, on a protein A-Sepharose column.

2. Isolation of cell populations.

 Human PBMCs were prepared by Ficoll-Isopaque density gradient centrifugation. The unfractionated population was separated into E rosette-less (E-) and E rosette-plus (Ec) populations by interaction with 5% sheep red blood cell (SRBC). The mixture was deposited on Ficoîl-Isopaque and the E cells were recovered from the interface while the E + cells were obtained from the pellet after hypotonic lysis of the SRBC.

The monocytes were obtained from E cells by adhering to the glass overnight. Peripheral blood mononuclear cells
CD2 + CD3 were obtained by complement-dependent lysis with a CD3 mAb in the E + cell fracture. Normal and leukemic samples were obtained from the Hospital Blood Bank
Saint-Louis and cryopreserved as previously described (23).

3. Cell cultures.

 Human T cell clones were obtained as described elsewhere (22) and cultured in RPMI-1640 medium (GIBCO, Paisley, Scotland) containing 2mmol / liter of L-glutamine, penicillin (100 U / ml) , streptomycin (100, ug / ml), 10% heat-inactive human serum and recombinant interleukin 2, kindly provided by Roussel-Uclaf (Romainville, France) (30 U / ml.) Cloned cells were restimulated every 7 days with feeder cells in the presence of purified PHA (Wellcome, Beckenham, UK) at 1, ug / ml and recombinant interleukin 2. The feeder cells were a mixture of irradiated allogenic LSPs from three donors (the same donors being used for several months). Cultures of EBV-transformed leukemia or B cell lines and hybridoma cell lines were mycoplasma free and maintained logarithmic in RPMI containing 10% selected heat-inactivated fetal calf serum and antibiotics.

4. Immunofluorescence studies.

 Indirect Immunofluorescence studies were performed using 1g antlsourls conjugated to fluorescein isothiocyanate (CITF) from Meloy Laboratories (Springfield, VA) as previously described (22). The fluorescence was read using a Facstar microfluorometer (Becton Dickinson, Mountain View, CA).

5. PMA-induced modulation of expression at the cell surface.

PBMCs were incubated with PMA (Sigma Chemical
Co., St. Louis, MO) to 1 ugiml in culture medium containing 1096 of
FCS during the Times indicated. After a complete wash, the cells were labeled with indirect fluorescence fluorescence using a battery of monoclonal antibodies against various surface molecules.

6. Labeling and immunoprecipitation of cell surface proteins.

 Cloned cells were labeled on the surface with lactoperoxidase and four additions of H2O2 as described above.

The cells were then lysed in a standard 10 mM Tris-HCl lysis buffer (pH 7.4), containing 1% (PV) of Nonidet P40, 0.15M NaCl, 1 mg / ml of BSA and 2 mM of PMSF (Phenyl Methyl Sulfonyl fluoride), 20 mM iodoacetamide, and aprotinin at 1 lU / ml as protease inhibitors. The immunopreclpltatlons and analysis by SDS-PAGE were performed as described (22). Non-reductive / reductive two-dimensional gel electrophoresis analyzes were performed using 7.5% acrylamide PHAST (Pharmacia) gels for the first and second dimensions according to the manufacturer's recommendations (Pharmacia, Uppsala, Sweden Autoradiography of dried gel plates was performed at -700C. Molecular weights were calculated by reference to the mobility of standard proteins.

7. Proliferation tests.

50,000 CMSP were cultured in triplicate in 96-well plates with rounded bottom (Costar, Cambridge, MA) in a total volume of 0.2 ml of RPM1 1640, supplemented with 10% fetal calf serum. Various concentrations of purified antibodies were added with 0.2 ng / ml PMA. After various periods of time, the wells were individually radiolabelled with 1.1 uCi (1 Ci = 37 Gbq) of (3H) Tdr and harvested 16 hours later. The incorporation of (3H) Tdr was measured using a scintillation counter for liquids (Rac Beta 1211/1212
LKB, Finland).

He. RESULTS 1. BB18 mAbs react strongly with cloned T cells and
weakly with human T cells at rest by cytofluorometry
flow.

 In order to obtain monoclonal antibodies defining the stages of T cell activation, six-week-old Balb / c mice were immunized with cloned human thymic cells (22).

An mAb of the lgGl isotype, named BB18, was thus isolated. Table I summarizes the reactivity of the abovementioned mAb with many normal and malignant hematopoietic human cells. The molecule recognized by this mAb is weakly expressed on resting T cells but not at all on B cells from peripheral blood or lymphoid organs. Among the leukemic lymphoid cell lines tested, all T cells were strongly labeled, while the Burkitt lymphoma cell lines were not reactive. Other malignant cell lines such as K 562 (erythroleukemia), U937 (monocytic),
HL60 (promeylocytic) or SKNSH (neuroblastoma) do not appear to be reactive.

In contrast, long-standing activated T cells (T cell clones
IL2-dependent) or B (EBV-transformed cell lines B) were found to be positive with this mAb. In addition, BB18 strongly labeled the YT cell line of activated NK, CD3 negative tumor cells and its mutant.
CD 25 negative, YT2C2. The structure recognized by BB18 appears to be strongly expressed immediately after cell activation as shown by the representative kinetic experiment carried out with peripheral blood mononuclear cells of a normal individual stimulated with PHA (Figure 1 ). The early increase in its expression occurred in parallel with that of C25 during the first days of activation of the T cell, but it remained persistent. A similar increase in reactivity to Bu 18 was also observed when peripheral blood lymphocytes (LSP) were stimulated with either allogenic cells, a CD3 mAb, or IL2 (data not shown). Therefore, the increased reactivity with Antibody BB18 clearly defines the activation state in T lymphocytes.

TABLE 1
Reactivity of the BB18 Antibody with populations of poetic blood cells
Type of cells% cells
positive
Normal cell population:
LSP E + (10) 100
LSP E (5) <5 PHA-activated lymphocytes (10) 100 cloned dependent IL2 T cells (10) 100 monocytes (5) <5 granulocytes (3) 100 red blood cells (3) <5 platelets (3) <5 thymus cells (3) 30 E + tonsil cells (2) 100 E tonsil cells (2) <s E spleen cells (2) 100b E spleen cells (2) transformed EBV cells (5) 100
Hematopoietic malignant cells
ECMT 100
MOLT4 100
YT2C2 100
Daudi <5
Namalva
NALM 6,100
U937 <5
HL60 <5
K562 <5
Non-hematopoietic malignant cells:
SKNSH (neuroblastoma)
U373MG (glioblastoma) <5 a Reactivity determined by flow microfluorometry b All cells were weakly labeled with mAb BBl8 2. The iritivity of reactivity with BB18 Antibody was reduced shortly after incubation with PMA.

As the expression of several structures inducible by activation is upregulated by PMA, we wanted to determine if the
PMA was able to rapidly increase reactivity with the BB18 Antibody. Surprisingly, after a 6 hour incubation with
PMA at 1, ug / ml, the labeling with the BB18 Antibody was no longer detectable.

As shown in the representative experiment presented in Figure 2, the reactivity of T cells to CD3, CD4 and Antibody Bu 18 decreases when PMA is added to the cultures. More precisely, after 6 hours with PMA, the down regulation of the molecule reacting with the Antibody BB18 is maximum while a large fraction of the cells had lost the expression of CD4. Modulation of the molecule
CD3 reaches its maximum after 24 hours while the modulation of CD4 is completed after 10 hours. On the other hand, as it was previously reported after 24 hours of culture in the presence of PMA, CD25 is induced and the expression of CD8 is not modified by this treatment.

3. Biochemical analysis of the immunoprecipitated molecule by mAb BB18
after labeling the cell surface.

In order to define the molecule identified by mAb BB18, human T cell clones were labeled with Iodine 125 on their external face, according to the lactoperoxidase method, and lysed. The BB18 immunoprecipitates obtained from these lysates of labeled cells were then analyzed by SDS-PAGE. The autoradiography shown in Figure 3 indicates that the AMC BB18 Immunoprecipitates from a representative T cell clone. A predominant band migrating under non-reducing conditions with a molecular mass of approximately 300 kDa. CD18 "M232" mAbs used as control immunopreclplt three bands of proteins from the same lysate characteristic of the CD18 molecules and two different molecules
CDII, while no protein band is obtained with an appropriate IgG1 type mAb. In addition, as shown in Figure 4, the immunoprecipitate obtained with Antibody BB18 consists mainly of homodimers linked by disulfide bridges of approximately 300 kDa in the first non-reducing dimension, which resolves in a second dimension reducing into a predominant protein band of approximately 150 kDa below the diagonal of the two-dimensional gel.

4. Stimulation with soluble BB18 mAbs induces proliferation of
LSP in the presence of a submitogenic concentration of PMA.

It has been shown previously that the stimulation of certain surface molecules, with their agonist antibody, in conjunction with
PMA, induces proliferation of T lymphocytes (24, 25, 26). Therefore, the possibility that cell proliferation could be induced by submitogenic doses of PMA and purified mAbs Bu 18 was tested. The results of a representative kinetic experiment, shown in FIG. 5, indicate that BB18 mAbs, used either at 5 or at 10 μg / ml with 0.2 ng / ml of PMA, induce a strong proliferative response on PBMCs freshly isolated reaching a peak on day 4. Under similar experimental conditions, a CD1 "L404" mAb of identical isotype has been shown to be incapable of triggering a proliferation of lymphocytes, whereas as previously reported, a CD3 mAb vigorously stimulates cell proliferation.

We therefore identified a new human lymphocyte cell surface structure with the mAb known as Bol8. The immunoprecipitated molecule using BB18, from lysates of donated T lymphocytes marked on the surface appeared to be a homodimer linked by disulfide bridges comprising subunits of approximately 150kDa. This structure is poorly detectable on resting peripheral blood T cells, while its expression increases rapidly after activation of the T cell and remains at this high level for some time after initial triggering. In addition, the Antibody
BB18 reacts strongly to all cloned T cells tested regardless of their CD4 / CD8 phenotype and their TcR (data not shown). Cell distribution indicates that Acm Bu 18 does not react with resting B cells, while cells B EBV-processed for a long time are weakly marked.

In contrast, Burkit lymphoma cell lines (maudi and
Namalva) clearly lack responsiveness. Furthermore, leukemic but not myelomonocytic lymphoid cell lines are reactive to BB18. Cellular distribution, as well as biochemical analysis of the structure recognized by BB18, have established that this molecule is different from all the surface structures of leukocytes previously known.

The functions of other surface molecules, such as LFA-l,
CD4 and CD8 have been demonstrated by the ability of certain specific mAbs to inhibit the natural or cytotoxic killer activities of T lymphocytes.

Attempts to inhibit, with a purified BB18 mAb, the cytotoxic function of allosensitized lymphocytes in vitro 6d., Cloned cytotoxic T lymphocytes or NK cells have not been successful.

The results suggest that the epitope recognized by mAb BB18 is not involved in cytotoxic functions, any more than in the adhesion of the effector to the target cell.

Of particular interest is the fact that CMSP cocultures with submitogenic concentrations of PMA and purified mAb BB18 resulted in a vigorous proliferative response, reaching a maximum on day 4. However, the comitogenic response measured under these experimental conditions does not reach the magnitude of the response observed with a CD3 trigger. It should be noted that the reactivities to CD3 and to BB18 both decrease after incubation of the LSPs with
PMA. However, the kinetics of the decrease in reactivity is different for the two structures.

 Several molecules have been described which, when reacted with an agonist mAb under appropriate conditions, are capable of transducing comitogenic signals with PMA. The molecule CD28, a 44 kDa disulfide-bridged homodimer, expressed preferentially on a T cell subpopulation, was the first structure described to induce costimulatory signals (25, 26). The effect has been shown to regulate the stability of the mRNA encoding lymphokines (27).

Other examples of costimulatory signal mediator molecules include the molecule CD26 (4, 5), identifying dipeptidylpeptidase IV, which is a 120 kDa structure; CD69, a very early activation heterodimer with disulfide bonds, of 28.32 kDa (17, 18); the T cell specific molecule identified by mAb lODI, which is a 90 kDa disulfide bridge homodimer (28); CD60 "UN14D4" recognizing a hydrocarbon structure expressed on various gangliosides (29). The CD43 mAbs are particularly interesting, identifying a highly sialylated glycoprotein of 95 kDa, which similarly to the CD3 and CD2 mAbs can directly trigger the activation of T cells in the presence of monocytes. It has recently been reported that a monoclonal antibody reactive with CD5, unlike the previously described CD5 mAb, was capable of inducing T cell proliferation in the presence of monocytes (15). This raised the possibility that only certain epitopes of the CD5 molecule, as in the case of CD2, are involved in the activation of T cells. Finally, it is worth mentioning that unlike the epitope recognized by the Antibody Bol8, the surface expression of the majority of signal transduction structures is not increased during activation of the cell.

Although human T cells can be activated through several distinct surface molecules, these activation pathways appear to share common signal transduction mechanisms. It is well known that activation of the T cell via CD2 or CD5, but not via CD43 (16) is dependent on the expression of CD3-TcR. Although no physical link of the BB18 molecule with CD3-TcR has been demonstrated according to the invention, (the mAb marked the YT2C2 lymphocytes
CD3-TcR negative), links are possible with components such as chain 4 of CD3.

ANTIBODY POT
Antibody-producing hybridoma cell lines
BD16 and BB18 have been deposited in the European Collection of Animal Cell
Cultures (Porton Down, Salisbury, Wiltstrive SP4 03G, England), January 7, 1992 and received the numbers 92010801 for the antibody BD16 and 92010802 for the antibody Bu 18.

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IV. W. Knapp Ed. Oxford University Press.

Claims (15)

 1. Antigenic protein consisting of a glycoprotein with a molecular weight of 150 + 10kDa determined by type electrophoresis SDS PAGE under reducing conditions, expressed on the surface of preferably activated human blood T lymphocytes.  2. Antigenic protein in the form of a disulfide-linked homo-dimer with a molecular weight of 300 + 20kDa determined by SDS PAGE type electrophoresis under non-reducing conditions, expressed on the surface of preferably activated human blood T lymphocytes, composed of subunits according to claim 1.  3. Derivative of a protein according to claim 1 or 2, characterized in that it is the protein in non-glycosilated form or comprising non-natural glycosilations or a fragment of the glycosilated or non-glycosilated protein comprising the antigenic sites. essentials.  4. Protein according to one of claims 1 to 3 characterized in that it comprises a marking allowing its assay in vitro or its location in vivo or ex-vivo.  5. Antibody directed against the protein according to one of claims 1 to 4.  6. Monoclonal antibody recognizing an epitope of a protein according to one of claims 1 to 4.  7. Antibody according to claim 5 or 6 characterized in that it is an immunoglobulin of subclass IgG1.  8. Antibody according to one of claims 5 to 7 characterized in that it is produced by a hybridoma cell line deposited at the ECACC under the number 92010801 for the antibody BD16.  9. Antibody according to one of claims 5 to 7 characterized in that it is produced by a hybridoma cell line deposited at the ECACC under the number 92010802 for the antibody BB18.  10. Antibody according to one of claims 5 to 9 characterized in that it comprises a marking allowing its assay in vitro or its location in vivo or ex-vivo.  11. Hybridoma cell line, characterized in that it is capable of producing the monoclonal antibody according to one of claims 5 to 10.  12. Use of one or two monoclonal antibodies according to claims 8 or 9 for immunoprecipitating proteins according to claims 1 or 2.  13. Diagnostic kit comprising a protein or one of the antibodies according to one of claims 1 to 10.  14. Use of a protein or an antibody according to one of claims 1 to 10 for the rapid diagnosis of a state of early activation of lymphocyte T cells.  15. Medicament comprising, as active principle, a protein or an antibody or both antibodies or their fragments according to one of claims 1 to 10.