WO2003062272A1 - Novel strategy for modulating t cell activation which is based on regulating the cd3ε - nck interaction - Google Patents

Abstract

The invention relates to the molecular mechanism for the activation of the T cells which takes place in inflammatory or immune reactions following activation from the binding of antigen/MHC to the TCR antigen receptor, demonstrating the key role of the specific interaction between determined domains of proteins CD3 epsilon and Nck in modulating the transmission of signals which occurs during said cell activation. Moreover, the invention relates to methods of modulating T cell activation using methods that allow the CD3 epsilon and Nck protein-protein interaction to be altered.

Description

NEW MODULATING STRATEGY FOR T-LYMPHOCYTE ACTIVATION BASED ON THE REGULATION OF CD3ε-Nck INTERACTION

TECHNICAL SECTOR

The present invention relates to the immune system and more specifically to the development of regulatory treatments for the T lymphocyte-mediated immune response in pathological processes. The development of these methods is based on the possibility of identifying new regulatory agents for T lymphocyte antigen receptor (TCR) function and on the use of new gene therapy strategies modulating intracellular signal transmission from said TCR.

STATE OF THE ART The cells respond to extracellular stimuli by means of receptors that cross the plasma membrane. Although some receptors are channels that allow ions to pass into the cytoplasm, most receptors do not transmit material through the membrane and depend on interaction with intracellular effector molecules to activate signal transmission pathways. In these cases, after ligand binding, surface receptors initiate signaling cascades within cells either after undergoing a conformational change or after forming aggregates (Bachmann and Ohashi, 1999; Cochran et al., 2001; Jiang and Hunter , 1999; Reth, 2001; Stoddard et al., 1992; Weiss and Schlessinger, 1998).

The receptor for T lymphocyte antigen (TCR) is responsible for the recognition of specific antigens bound to the major histocompatibility complex (MHC) present in so-called antigen presenting cells (APC). At the T lymphocyte interface: APC, the two membranes form a structure known as the immune synapse (IS) (reviewed in (Bromley et al., 2001; Dustin et al, 1998). The TCR is concentrated in the IS forming a Supramolecular activation aggregate (SMAC) surrounded by a ring of adhesion molecules (Monks et al, 1998). The formation of IS requires signals from the TCR as well as rearrangement of the actin cytoskeleton and is believed to be necessary for the TCR give a sustained signal leading to activation of T lymphocytes. TCR is composed of a TCRα / β heterodimer (or TCRγ / δ in T γδ lymphocytes) bound to a set of four dimer-organized polypeptides: the CD3γ-CD3ε and CD3δ-CD3ε heterodimers. and the CD3ζ-CD3ζ homodimer (Clevers et al., 1988). TCRα / β recognizes the antigen / MHC complex and somehow transmits this information to CD3 components. All TCR subunits are type I membrane proteins, but unlike the TCRα / β heterodimer, CD3 components have cytoplasmic stems that can interact with intracellular signal transmission molecules. The CD3 subunits each have one (CD3γ, CD3δ and CD3ε) or three (CD3ζ) tyrosine and leucine motifs, called tyrosine-based activation motifs present in immunoreceptors (ITAM) (Reth, 1989). Once they are phosphorylated by the src family tyrosine kinases, tyrosine residues within these motifs become binding sites for proteins with src type 2 (SH2) homology domains, such as ZAP70 tyrosine kinase ( reviewed in (Kane et al., 2000; Lin and Weiss, 2001; Qian et al., 1997). ITAM phosphorylation is believed to be the earliest activation event that occurs after ligand binding to TCR. On the other hand, although the entire cytoplasmic stem of the CD3 subunits has been conserved throughout evolution, to date no role in activation has been described for sequences located outside ITAMs.

There are several methods described to regulate the function of lymphocytes. Since T lymphocytes play a critical role in regulating the immune response, the use of drugs that block the activation of T lymphocytes has become desirable for the prevention of organ allograft rejection and for the control of diseases of etiology. autoimmune: rheumatoid arthritis, lupus erythematosus, multiple sclerosis, type I diabetes, myasthenia gravis, to name a few, and allergic type. On the other hand, it is also convenient to control the expansion of T lymphocytes in the case of T-cell lymphomas.

Currently, the most widely used drugs for the prevention of allogeneic transplant rejection are cyclosporin A and FK506; both serine phosphatase calcineurin inhibitors. However, the chronic use of both inhibitors poses serious nephrotoxicity and neurotoxicity problems, possibly due to the fact that calcineurin, although it plays a fundamental role in the activation of T lymphocytes, is expressed in other tissues where it also plays important roles. A immunosuppressive therapy based on the use of polyclonal or monoclonal anti-lymphocyte antibodies. These treatments have been shown to be effective in preventing organ rejection, but they also suffer from problems of toxicity and the generation of resistance to treatment mechanisms in the patient that prevent their chronic use. For all these reasons, it is necessary to discover new agents that allow a selective inhibition of the activation of T lymphocytes and that therefore do not interfere with the functions of other tissues. The specific activation pathways of T lymphocytes can be perfect targets for new and selective immunosuppressive agents and therapies. On the other hand, current therapies for stimulating T lymphocytes (in infections and tumors) are often insufficient to induce an adequate immune response, so that new strategies would allow expanding the efficacy of said treatments.

DESCRIPTION OF THE INVENTION

Definitions: a) Homologous forms of the Nck and CD3ε proteins, as used in the present invention, indicate to all those proteins that, with respect to these and regardless of their origin, have a homology of at least 30%, preferably at least 85%, or more preferably at least 95%. b) Nck, the NcK protein as used as a general reference in the present invention comes to indicate both the NcKα protein and the NcKβ protein.

Evidence of the molecular mechanism of T lymphocyte activation that occurs in inflammatory or immune reactions following activation from antigen / MHC binding to the TCR antigen receptor is provided in the present invention, demonstrating the key role of the specific interaction between certain domains of CD3ε and Nck proteins in the modulation of signal transmission that takes place in said activation of lymphocytes.

The present invention provides methods for the modulation of inflammatory or immune T lymphocyte activation dependent on the CD3ε and Nck interaction by methods that allow the alteration of the protein-protein interaction of CD3ε and Nck.

A specific object of the present invention is the modulation of said CD3ε-Nck interaction through the use of chemical substances that alter said interaction. If that such chemicals block the CD3ε-Nck interaction will be identified as substances that inhibit (antagonize) the immune or inflammatory response, whereas if they activate (agonists) said CD3ε-Nck interaction, substances that activate these immune or inflammatory responses will be identified. A specific object of the present invention is the use of said substances that antagonize T lymphocyte activation in treatment protocols for diseases that cause pathological activation of T lymphocytes such as, among others, autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, multiple sclerosis, type I diabetes and myasthenia gravis; allergic-type diseases; in the prevention and treatment of transplant rejection and T lymphocyte lymphomas. A specific object of the present invention is the use of said T lymphocyte activation agonist substances in protocols for the treatment of diseases that have an inhibition of T lymphocytes such as, among others, infectious, bacterial and viral diseases, among others, and tumors.

Another object of the present invention is a method for the identification of said agonist and antagonist substances of the cellular function of the protein-protein CD3ε-Nck interaction that can occur in physiological and pathological processes, among others, inflammatory, autoimmune, allergic, infectious and tumorous. A specific object of the present invention is a method for the identification of agonists and antagonists of the activation of T lymphocytes mediated by the interaction of CD3ε-Nck proteins, which comprises the following steps: a) generating a biological preparation of the protein interaction -protein that mimics the CD3ε-Nck interaction, b) adding the candidate chemical compound agonist or antagonist, and c) determining the blockade or activation of the protein-protein CD3ε-Nck interaction, identifying in the case of blocking an antagonist of the CD3ε interaction- Nck and in case of activation an agonist of the interaction

CD3ε-Nck.

Another object of the present invention is the previous method of identifying agonists and antagonists of the CD3ε-Nck interaction characterized in that the biological preparation of the protein-protein interaction of CD3ε-Nck is carried out using the human CD3ε and Nck proteins, their homologous forms. or peptides

(fragments) that mimic the biological activity of these proteins or their forms homologous. Another specific object of the present invention is a peptide that mimics the biological activity of CD3ε-binding NcK characterized by the amino acid sequence of the SH3.1 domain of NcKβ (SEQ ID NO2). Another specific object of the present invention is a peptide that mimics the biological activity of NcK binding to CD3ε characterized by the amino acid sequence of the SH3.1 domain of NcKα (SEQ ID NO3) as an element of said biological preparation. Another specific object of the present invention is a peptide that mimics the biological activity of Nck-binding CD3ε characterized by the amino acid sequence of the proline rich region (PRS) of CD3ε described in the present invention (SEQ ID NO1) as an element of said biological preparation. Another object of the present invention is a fusion protein that comprises, in addition to the amino acid sequence of human CD3ε and Nck proteins, their homologous forms or peptides (fragments) that mimic the biological activity of said proteins or their homologous forms, other amino acid sequences that facilitate the subsequent determination of the alteration of the CD3ε-Nck interaction as an element of said biological preparation (among others and as an example: GST-SH3.1, SH3.1-EGFP, Nck-EGFP and HA-Nck) . Another object of the present invention is the previous method of identifying agonists and antagonists of the CD3ε-Nck interaction characterized in that the biological preparation of the protein-protein interaction of CD3ε-Nck is carried out using the human CD3ε and Nck proteins, their homologous forms. or peptides (fragments) that mimic the biological activity of said proteins or their homologous forms derived from used cells or from preparations of purified or semi-purified proteins.

Another specific object of the present invention is a method of identifying agonists and antagonists of the CD3ε-Nck interaction characterized in that the biological preparation of the protein-protein interaction of CD3ε-Nck is carried out in a cell-free environment (among other possibilities, the immunoprecipitation assays carried out in example 1 and the stimulation assay of the purified TCR with the anti-CD3 antibody UCHT1 and subsequent blocking with the APAl / 1 antibody, see example 4). The protein-protein complexes that mimic the CD3ε-Nck interaction and that are part of the procedure for identifying agonists and antagonists of the CD3ε-Nck interaction can be identified and quantified using a wide range of protein labeling techniques, among others, by immunological techniques, as performed in the present invention (anti-Flag and anti HA).

Another object of the present invention is a method of identifying agonists and antagonists of the CD3ε-Nck interaction characterized in that the biological preparation of the protein-protein interaction of CD3ε-Nck is carried out in a cellular environment, among other possibilities: a) assay functional quantification of cell extension and deployment, actin cytoskeleton polymerization, (see example 5), b) functional assay for quantification of cytokine secretion, IL-2 and TNFα (see example 5), c) functional assay for quantification of cell proliferation (see example 5), d) functional assay to quantify the formation of conjugates between lymphocytes and APC cells (see example 6), and e) functional assay to quantify IS maturation (see example 6). Another object of the present invention is a method of identifying agonists and antagonists of the CD3ε-Nck interaction characterized in that the biological preparation of the protein-protein interaction of CD3ε-Nck is carried out in a cellular environment, consisting of a two-hybrid assay (trap assay) (for a more detailed description of this type of assay as well as other examples of assays aimed at identifying compounds modulating protein interactions see: United States Patent 6,037,136 Beach, et al., March 14, 2000 Interactions between RaF proto-oncogenes and CDC25 phosphatases, and uses related thereto and United States Patent 5,723,436 Huang, et al, March 3, 1998 Calcineurin interacting protein compositions and methods). Another object of the present invention is the previous method of identifying agonists and antagonists of the CD3ε-Nck interaction characterized in that the biological preparation of the protein-protein interaction of CD3ε-Nck and the determination of the blocking or activation of said interaction after the addition of an agonist or antagonist candidate is performed in a basal or control situation, of stimulation (anti-CD3 and APC cells loaded with superantigen or antigen, among others) or blocking (overexpression of SH3.1 of Nck and transduction of APA1 antibody / 1, among others) of said interaction according to the interest in identifying one or the other agent. So an example A specific test to identify a possible antagonist of the Nck-CD3ε interaction could consist of the tests carried out in example 5 or 6 of the present invention, where an antagonist would produce a block of the activation obtained after stimulation of the TCR complex either with the anti CD3 antibody UCHT1 or with APCs loaded with SEE both in the control situation (EGFP) and in the case of cells transfected with Nck-EGFP. And a concrete example of an assay to identify an agonist of the Nck-CD3ε interaction could consist of the assays performed in Example 6 of the present invention where an agonist would produce a reversal of the blockade of the TCR complex obtained by the overexpression of the SH3 domain .1 of Nck both in the control situation (EGFP) and in the case of cells transfected with Nck-EGFP. Finally, there are many other types of tests, and that with the information provided by the present patent, may be applicable thanks to the knowledge existing in the state of the art and are part of the present invention. Chemical compounds that are candidates for testing as modulators of the CD3ε-Nck interaction can be produced, for example, by bacteria, yeasts, and other microorganisms (natural products), chemically produced (for example, small molecules including peptidomimetics), or produced by genetic engineering.

Another object of the present invention are methods for modulating the CD3ε and Nck interaction by means of gene manipulation that allow blocking of the protein-protein interaction of CD3ε and Nck and therefore the implantation of new gene therapy treatments for diseases in which it is necessary to block the activation of T lymphocytes such as, among others, autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, multiple sclerosis, type I diabetes and myasthenia gravis, allergic-type diseases, in the treatment and prevention of transplant rejection and treatment of T lymphocyte lymphomas. Thus, a specific object of the present invention is a treatment of gene therapy of diseases that occur with an activation of T lymphocytes characterized in that the blocking of the CD3ε-Nck interaction is carried out, among others, by overexpression of the SH3.1 domain of Nck or by transduction with a specific antibody ífico (APA1 / 1). In this method, a nucleotide encoding the SH3.1 domain of Nck, the Nck protein, its homologous forms, or any other peptide (fragment) that mimics said functional activity of interaction with the PRS domain of CD3ε is inserted into a suitable vector, by example a plasmid. Nucleic acid can be genomic DNA, cDNA or RNA. Said DNA or RNA can be isolated and integrated into a vector by standard methods known in the state of the art. Such methods are described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory 1989). Another specific object of the present invention are the nucleotides (DNA, RNA or cDNA) that encode human CD3ε and Nck proteins, their homologous forms or peptides (fragments) that mimic the biological activity of said proteins or their homologous forms and that they form part of any of the above methods of modulating the CD3ε and Nck interaction. Furthermore, those vectors that comprise the above nucleotides as well as all those host cells that contain said vectors are part of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1.- Nck is recruited to CD3ε after binding of ligands to TCR. (A) Nck binds to CD3ε but not to the other CD3 subunits. COS cells transfected with the indicated constructs were used and precipitation (pd) was performed with the GST-Nck fusion protein followed by immunoblotting with anti-Flag or anti-CD3ζ antibodies. A fraction of the total lysate from each transfectant was hybridized with the corresponding antibody to demonstrate the presence of the protein. (B) Nck is recruited to the TCR after stimulation of intact T lymphocytes. Jurkat cells were stimulated for 5 min (time 5) with 10 µg / ml of the anti-CD3 antibody UCHT1 or incubated without antibody (time 0). The TCR was immunoprecipitated with the anti-CD3ε SP34 antibody and immunoblotting was performed with an anti-Nck antibody. A fraction of the total lysate (TL) from unstimulated cells was run in parallel to indicate the position of Nck. (C) Transfected Nck binds to endogenous TCR upon activation. Jurkat cells were transfected, or not transfected, with a vector expressing Nckβ labeled with an HA epitope. Immunoprecipitation with UCHT1 and immunoblotting with anti-HA was performed. Proteins running below the HA-Nckβ position (arrow) probably correspond to fragments of the antibodies used for stimulation and immunoprecipitation. (D) Nck constitutively binds to immobilized CD3ε. GSTε precipitation was performed from Jurkat cells transfected with HA-Nckβ followed by immunoblotting with anti-HA. (E) Stimulating the TCR with Antibody induces its association to immobilized Nck. Jurkat cells transfected with Flag-CD3ε were stimulated with UCHTl. The TCR was precipitated with GST-Nckβ and revealed by immunoblotting with anti-Flag and anti-CD3ζ. A GST precipitation from used cells from stimulated cells was run in parallel as a negative control. (F) Antibody stimulation promotes binding of TCR to Nck in untransformed T lymphocytes. Human PBMC (peripheral blood mononuclear cells) were stimulated with UCHTl and binding of TCR to Nck was demonstrated after precipitation with GST-Nckβ and immunoblotting with anti-CD3ζ. (G) Antigen stimulation promotes recruitment of Nck to TCR in isolated thymus and mouse spleen T lymphocytes. Thymocytes and spleen cells from mice transgenic for the TCR AND were stimulated (+) with DCEK APC previously loaded with PCC antigen. Immunoprecipitation was performed with the anti-TCRβ H57 antibody and immunoblot with anti-Nck. The filter was subsequently rehybridized with anti-CD3ζ as charge control. Figure 2.- The amino-terminal SH3 domain of Nck binds to the proline-rich sequence of CD3ε.

(A) Map of the deletions made in the cytoplasmic stem of human CD3ε. Comparison of the amino acid sequence of the cytoplasmic stem of the CD3ε chain of different species. The conserved amino acids are shown in normal type while the non conserved ones are shown in gray. Amino acids corresponding to PRS or ITAM are shown in bold. Deleted sequences in human CD3ε mutants are indicated with a dashed line and corresponding number. (B) Characterization of the Nck binding sequence in CD3ε. COS cells were transfected with wild-type CD3ε (wt) or with the indicated deletion mutants (d7 to di 6) or were left without transfection (nt). GST-Nckβ precipitation was performed after labeling the cells with ³⁵ S-methionine. Phosphorimager images are shown (upper panels). Immunoprecipitation with anti-CD3ε SP34 antibody was performed as a control of transfection (lower panels). (C) Nckα and Nckβ can be associated with TCR. Sepharose bead-coupled GST-Nckα and GST-Nckβ were used to precipitate the TCR of Used from Jurkat cells not stimulated or stimulated with UCHT1. (D) Identification of the TCR binding site in Nck. The indicated GST fusion proteins were used to precipitate the TCR, revealed by immunoblot with anti-CD3ζ antibody. Nckβtrn is a Nck mutant lacking the SH2 domain.

Figure 3.- Induction of TCR binding to Nck is independent of tyrosine kinase activation and occurs before protein phosphorylation in tyrosine is detected.

(A) Binding of TCR to Nck is independent of tyrosine kinase activity. GST-SH3.1 was used to precipitate the TCR from Jurkat cells stimulated for 5 min with UCHTl, stimulated with pervanadate (PV) or not stimulated. An immunoblot was performed with the anti-CD3ζ antibody (upper left panel). As control of tyrosine kinase activation, immunoprecipitation and immunoblotting with the anti-phosphotyrosine antibody 4G10 was performed from the same Usages (lower left panel). To study the effect of PP2 tyrosine kinase inhibitor on TCR-Nck interaction, Jurkat cells were stimulated 5 min with UCHTl in the presence (5 + PP2) or in the absence (5) of 20 μM PP2, and the TCR-Nck association was revealed as above (upper right panel). The effect of PP2 treatment on tyrosine kinase activity was demonstrated by immunoprecipitation and immunoblotting with 4G10 (lower right panel). (B) TCR binding to Nck is detected before protein phosphorylation in tyrosine. Jurkat cells were stimulated with UCHTl for the indicated times before precipitation with GST-Nckβ and immunoblot with anti-CD3ζ (upper panel). Immunoprecipitation and immunoblotting with 4G10 was performed in parallel to show tyrosine phosphorylation of total proteins (intermediate panel), while immunoprecipitation with UCHTl and immunoblotting with 4G10 was performed to show tyrosine phosphorylated CD3ζ (lower panel). (C) Temperature dependence of TCR to Nck binding. Jurkat cells were stimulated with UCHTl for 5 min at the indicated temperatures and treated as above to show the association of TCR to Nck (upper panel) and the level of tyrosine phosphorylation of total proteins (lower panel). Figure 4.- The binding of a stimulating antibody to the purified TCR induces a conformational change that allows the binding of the TCR to Nck. (A) Stimulation of TCR with purified antibody induces CD3ε binding to Nck. The TCR complex of clone 31.13 scVβ3 expressing TCR-Fv was purified on NP-Sepharose columns and eluted with free NJP. The purified TCR was subsequently incubated with 50 ng / ml of the UCHT1 stimulating antibody, and subsequently with 1 µg / ml of the anti-CD3ε APA1 / 1 antibody or with 1 µg / ml of the control anti-CD3δ APA1 / 2 antibody before precipitation of the TCR with GST-SH3.1. (B) A monovalent Fab fragment of the anti-TCR stimulating antibody induces the conformational change in the TCR that promotes Nck binding. TCR purified from 31.13 scVβ3 cells was incubated with 50 ng / ml UCHtl or 50 ng / ml of its Fab fragment before precipitation with GST-SH3.1. (C) Model of T cell activation by the TCR. The binding of the antigen / MHC to the variable regions of the TCRα / β heterodimer promotes both aggregation and a conformational change in the TCR. TCR aggregation would be necessary for activation of TCR-associated Lk and Fyn kinases from the src family, which subsequently phosphorylate ITAMs. Independently, a conformational change occurs in the cytoplasmic stems of the CD3 subunits resulting in exposure of the proline-rich sequence of CD3ε (indicated as a P in the Figure) and in Nck recruitment. Located at a second level would be the recruitment of ZAP70 tyrosine kinase and other signaling molecules to phosphorylated ITAMs and the recruitment of WASP, WD ?, SLP76 and Pak to Nck. A dimeric model of the TCR is shown (Fernandez-Miguel et al., 1999; San José et al, 1998). Figure 5.- Interference with the Nck-CD3ε interaction inhibits the activation of T lymphocytes. (A) Overexpression of the SH3 domain binding to Nck CD3ε inhibits actin polymerization and cell extension. Stable Jurkat cell transfectants expressing forms fused to complete Nck EGFP (Nck-EGFP) or the SH3.1 domain of Nck (SH3.1-EGFP) or the empty vector (EGFP) were stimulated for 10 min on coverslips coated with 25 μg / ml UCHTl (anti-CD3) or poly-Usin (poly-D-Lys). Cells were fixed and stained with Texas Red labeled phalloidin to show the presence of F-actin. The percentage of spread cells was estimated in the fluorescence microscope and was 87% for cells transfected with EGFP, 91% for cells transfected with Nck-EGFP and 2% for cells transfected with SH3.1-EGFP. (B) Overexpression of the N3 SH3 domain that binds to CD3ε inhibits the release of IL-2 produced by anti-CD3 (black column). Jurkat cells expressing the indicated constructs were stimulated for 24 h in 96-well plates coated with anti-CD3. After stimulation, the supernatant was collected and the amount of IL-2 was measured. (C and D) Effect of SH3.1 overexpression on superantigen-induced cytokine release. Jurkat cells expressing the constructs Nck-EGFP (triangles), SH3.1-EGFP (circles) and EGFP (squares) were stimulated for 24 h with Raji cells preincubated with superantigen (SEE). After stimulation, the supernatant was collected to measure the release of IL-2 and TNF-α. (E) Interference with Nck binding to CD3ε inhibits the proliferation of T lymphocytes. Human PBMC were transduced with APAl / 1 (circles) or with anti-CD4 antibody HP2 / 6 (squares) and stimulated with UCHTl immobilized on plastic. 48 h later, proliferation was estimated by incorporation of ³ H-thymidine. The arithmetic mean and standard deviation of a quadruplicate experiment are represented. (F) Transduction with the APA1 / 1 antibody interferes with the binding of CD3ε to Nck. Human PBMC were transduced or not with the APA1 / 1 antibody and stimulated for 5 minutes with UCHTl. GST-SH3.1 precipitation was performed followed by immunoblotting with anti-CD3 evaluar to assess binding of TCR to Nck. A control immunoprecipitation with 4G10 followed by immunoblotting with anti-CD3ζ was performed to assess the level of phospho-CD3ζ. Figure 6.- Interference with the Nck-CD3ε interaction inhibits the formation of T lymphocyte conjugates with APC and the maturation of the immune synapse.

(A) CMAC-labeled Raji cells were incubated with 5 µg / ml SEE and mixed with Jurkat cells in a 1: 1 ratio. The conjugates were adhered to poly-Usin coated coverslips and counted by direct examination under the fluorescence microscope. The bars represent the percentage of conjugates relative to the total number of Raji cells. The arithmetic mean + standard error of six experiments is shown.

(B) Raji cells were labeled with CMTMR and subsequently with SEE and deposited on fibronectin coated coverslips. Jurkat cells transfected with Nck-EGFP were added and the individual cells were followed by confocal video microscopy. Images were taken at 30 "intervals and representative images taken from digital video at the indicated times are shown. Each frame represents the DIC image superimposed with the fluorescence images of Raji cells (red) and Nck-EGFP (green). (C ) Jurkat and Raji cell conjugates were formed as described in (A), fixed and stained to show TCR or PKCΘ in red, images on the right, corresponding DIC images superimposed on staining are shown on the left in Raji cell blue. (D) The percentage of cumulative PKCΘ conjugates was determined. in the contact area relative to the total number of conjugates. The arithmetic mean + standard error of four independent experiments is shown.

EXAMPLES OF THE INVENTION In the present invention it is described that NcKβ, an adapter protein involved in the regulation of the actin cytoskeleton promoted by receptor binding (Buday, 1999; Li and She, 2000; Myung et al, 2000), interacts with the cytoplasmic stem of CD3ε specifically and that this interaction induces a modification in the TCR receptor that allows the binding of the entire TCR complex to Nck and, therefore, the activation of T lymphocytes. On the other hand, in the present invention it has been verified that this interaction also occurs in different types of T lymphocytes after being stimulated: Jurkat human T lymphocyte cell line,

Peripheral blood human T lymphocytes and mouse lymphocytes from thymus and spleen stimulated with anti-CD3 antibodies, and - Thymic and spleen T lymphocytes from mice transgenic for TCR AND stimulated with APCs loaded with specific antigen; which has shown that stimulation of the TCR with antigen / MHC or with antibodies causes a modification of the TCR that allows the recruitment of NcK to CD3ε (Example 1). On the other hand, and for the first time, the regions involved in both proteins, NcK and CD3ε, have been identified at the molecular level in said interaction. Thus, a proline-rich sequence (PRS) of the cytoplasmic domain of CD3ε has been identified, which is absent in the other CD3 subunits, and which has been identified in the present invention as being present within the region of CD3ε deleted in the mutants 9 and 10 (Figure 2C) (SEQ ID NO 1) as directly related to the interaction with NcKβ (Example 2). On the other hand, the amino-terminal SH3 domain (SH3-1) of NcKβ (SEQ ID NO 2) has been identified as the region of this protein involved in interaction with CD3ε (Example 2). It should be noted that two human genes corresponding to NcK have been described that share 67% homology (Buday, 1997). These two genes have an identical structure in three SH3 domains and one C-terminal SH2 domain. Although the form initially identified in the two-hybrid assay (Example 1) was NcKβ, it has been found in the present invention that the two forms bind comparably to TCR as demonstrated in precipitation experiments with GST-NcKα and GST-NcKβ. Consequently, constructs of all or part of NcKα and NcKβ were used interchangeably in the subsequent experiments of the present invention. The SH3.1 region of NcKα is described in SEQ ID NO 3. The fact that the interaction between Nck and CD3ε was mediated by an interaction

PRS-SH3 made it difficult to explain how Nck recruitment to TCR could be inducible. Since the PRS in CD3ε is located near ITAM, one possible explanation was that tyrosine phosphorylation in some way influenced the accessibility of PRS to Nck. However, in the present invention it could be concluded that the change in TCR after binding of Nck to TCR is independent of PTK activity and precedes ITAM phosphorylation in tyrosine (Example 3).

Subsequently, in the present invention, it was verified that the stimulation of the TCR in living lymphocytes by ligand binding promotes a conformational change that results in the exposure of the PRS region of CD3ε and its interaction and binding with the SH3.1 region of NcK ( Example 4). Ligand binding to TCR would cause a readaptation of the CD3ε tail in a structure that would allow exposure of the proline rich region (PRS), whereas in a situation of lack of stimulation of the TCR, the CD3ε tail would present a structure that would prevent binding with NcK.

Furthermore, it has been found that blocking the site of interaction with NcK in CD3ε by overexpression of the Nck SH3.1 domain or by transduction with a specific antibody (APA1 / 1) causes the inhibition of activation of T lymphocytes (Example 5). Finally, it has been found that this inhibition of TCR-mediated activation of T lymphocytes obtained by overexpression of the CD3ε-binding NcK domain is produced by a reduction in the number of T: APC conjugates formed and by an inhibition of the maturation of the IS (Example 6).

In summary, the characterization of the NcK-CD3ε interaction described for the first time in the present invention identifies NcK as an immediate effector of the TCR framed in the following model: The binding of the antigen / MHC to the variable regions of the TCRα / β heterodimer promotes at the same time the aggregation and a conformational change in the TCR. TCR aggregation would be necessary for activation of TCR-associated Lk and Fyn kinases from the src family, which subsequently phosphorylate ITAMs. Independently, a conformational change occurs in the stems cytoplasmic subunits of CD3 resulting in exposure of the proline-rich sequence of CD3ε (indicated as a P in Figure 4C) and in Nck recruitment. Located at a second level would be the recruitment of ZAP70 tyrosine kinase and other signaling molecules to the phosphorylated ITAMs. Also at this level would be the recruitment to Nck of other proteins through domains other than SH3.1. These proteins are: WASP (third domain SH3), WD? (second SH3 domain), SLP76 (SH2 domain) and Pakl (second SH3 domain) (Buday, 1999; Li and She, 2000). Therefore, Nck has the potential to simultaneously bind to TCR and other important proteins involved in actin cytoskeleton polymerization and signal transduction, and thus can become a modulating site for T cell activation.

The following Examples serve to illustrate the invention and should not be construed as limiting the scope of the invention. Example 1.- Nck binds to CD3ε after the interaction of the TCR with its ligands. Using the yeast two-hybrid system based on recruitment of

SOS (Aronheim, 2001) determined that Nckβ interacts with the cytoplasmic stem of CD3ε. Nckβ specifically bound to a bait construct consisting of two tandem copies of the cytoplasmic stem of CD3ε, and also to a single copy (Table 1). To check in another system that Nck binds to CD3ε and if it binds to other CD3 subunits, we used a GST-Nckβ fusion protein to precipitate from Used COS cells transfected with the different CD3 subunits. We found that GST-Nckβ bound CD3ε but not CD3γ, nor CD3δ, nor CD3ζ, indicating that CD3ε is the only subunit of the CD3 complex that interacts with Nckβ (Figure 1A). To check whether Nck and CD3ε interact in T lymphocytes, immunoprecipitation with anti-CD3ε was performed from the human T cell line Jurkat, followed by immunoblotting with anti-Nck (Figure IB). Nck was co-precipitated together with CD3ε from Jurkat cells stimulated with the anti-CD3 antibody UCHTl for 5 min, but not from unstimulated cells. To verify that the protein band detected by blot represents Nck, Jurkat cells were transiently transfected with Nckβ labeled with an HA epitope (Figure 1C). Immunoblotting with anti-HA confirmed the presence of Nckβ in CD3ε immunoprecipitates only when starting from cells transfected with HA-Nckβ (Figure 1C). Again, the association of Nck to CD3ε was induced by stimulation with anti-CD3. Binding of Nck to CD3ε dependent on stimulation might be necessary for a modification of Nck or the TCR itself. To check which of these two possibilities is true, we did precipitation experiments using GST fusion proteins. On the one hand, we verified that Nck associates with a fusion protein consisting of GST bound to the cytoplasmic stem of CD3ε independently of stimulation (Figure ID). For the reciprocal experiment, Jurkat cells transfected with CD3ε labeled with a flag epitope were used. Precipitation with GST-Nckβ followed by immunoblotting with anti-flag showed that the association of endogenous CD3ε with immobilized Nck is stimulation dependent (Figure 1E). In the same experiment, we found that binding of TCR to Nck could also be followed by immunoblotting with an anti-CD3ζ antibody. Since CD3ζ is the last subunit to assemble in the TCR (for review see (Jacobs, 1997), these results suggest that stimulation with anti-CD3 induces a modification in the TCR that allows the binding of the entire TCR complex to Nck .

To verify whether Nck recruitment could be replicated with more physiological stimuli and on different T lymphocytes, a number of different systems were used. First, the association of CD3ε with Nck was induced after anti-CD3 stimulation of peripheral blood human T lymphocytes (Figure 1F). In the same way, stimulation of T lymphocytes from thymus and mouse spleen with anti-CD3 antibodies promoted the binding of CD3ε to GST-Nckβ (data not shown). Finally, the association of TCR with Nck was also detected when thymic and spleen T lymphocytes from mice transgenic for the TCR AND were stimulated with APCs loaded with antigen specific to that TCR (Figure 1G). This demonstrates that stimulation of the TCR with antigen / MHC also induces the recruitment of endogenous Nck. Taken together, these results indicate that stimulation of the TCR with antibodies or antigen results in a modification of the TCR that allows the recruitment of Nck to CD3ε. Example 2.- The amino-terminal SH3 domain of Nck binds to a proline-rich sequence in CD3ε. To understand the molecular basis of the dependence of the Nck-interaction

CD3ε of TCR stimulation, a series of precipitation experiments were performed to identify the responsible regions in both CD3ε and Nck. A set of deletion mutants comprising the entire cytoplasmic region of CD3ε (Figure 2A) was transfected into COS cells. After metabolic labeling with ³⁵ S-methionine, cells were used and GST-Nckβ was used for precipitation assays. These experiments demonstrated that Nckβ interacts with wild-type CD3ε and also with mutants 7, 8, 11, and 12 but not with mutants 9 and 10 (Figure 2B, upper panel). A proline-rich sequence (PRS) that is absent in the other CD3 subunits, is contained within the CD3ε region deleted in mutants 9 and 10 (Figure 2A). Deletion of this sequence prevents binding to Nck. Two human genes corresponding to Nck have been described that share 67% homology (Buday, 1999). These have an identical organization in three SH3 domains and one C-terminal SH2 domain. Although the form we initially identified in the two-hybrid assay was Nckβ, in reality the two forms bind comparably to TCR as demonstrated in precipitation experiments with GST-Nckα and GST-Nckβ (Figure 2C). Consequently, constructs of all or part of Nckα and Nckβ were used interchangeably for the following experiments. Since Nck contains three SH3 domains, it seemed feasible that the Nck-CD3ε interaction was mediated by the binding of one or more of these SH3 domains to the CD3ε PRS. Accordingly, a truncated form of Nck lacking the SH2 domain bound to CD3ε in the TCR inducibly, while GST bound to the SH2 domain did not precipitate the TCR (Figure 2D). Using GST constructs bound to individual SH3 domains, we found that the amino-terminal SH3 domain (SH3.1) of Nck, but not others, binds to CD3ε (Figure 2D). This binding was also inducible, indicating that the SH3.1 domain is responsible for the binding of Nck to CD3ε. As a control of specificity, we verified that the second SH3 domain of Nck binds to Cbl constitutively (data not shown), as previously described (Wunderlich et al., 1999).

Example 3.- The change in the TCR that promotes its binding to Nck occurs before and independently of protein phosphorylation in tyrosine.

The fact that the interaction between Nck and CD3ε was mediated by a PRS-SH3 interaction made it difficult to explain how the recruitment of Nck to TCR could be inducible. Since the PRS on CD3ε is located near ITAM, one possible explanation was that tyrosine phosphorylation somehow influenced the accessibility of PRS for Nck. This hypothesis was tested using inhibitors and activators of tyrosine kinases. First, Jurkat cells were stimulated with pervanadate (PV), a treatment that increases the level of tyrosine phosphorylation and induces a series of activation processes similar to those stimulated by TCR (Secrist et al., 1993). Unlike stimulation with anti-CD3 antibodies, stimulation with PV did not promote the association of TCR with GST-SH3.1 (Figure 3A, upper left panel). As control that the PV treatment had been effective, we performed immunoprecipitation and immunoblotting with an anti-phosphotyrosine antibody (Figure 3A, lower left panel). Second, Jurkat cells were incubated with PP2, a very potent src family tyrosine kinase (PTK) inhibitor (Hanke et al., 1996) such as Lck and Fyn. These PTKs are believed to be responsible for the initiation of the activation cascade by promoting tyrosine phosphorylation within ITAMs (Lin and Weiss, 2001). The addition of PP2 completely prevented anti-CD3-stimulated tyrosine phosphorylation (Figure 3A, lower right panel), while the association of TCR with Nck was unaffected (Figure 3A, upper right panel). Thus, Nck binding to TCR appeared to involve changes in TCR independent of PTK activity.

The phosphorylation of ITAMs in tyrosine is believed to be the earliest event induced after TCR stimulation (Lin and Weiss, 2001). Since Nck binding is independent of tyrosine phosphorylation, we study the time course of both processes. TCR stimulation for a time as short as 5 seconds induced its association with Nck (Figure 3B, upper panel), while the level of tyrosine phosphorylation did not differ from that of unstimulated cells (Figure 3B, intermediate panel). Furthermore, CD3ζ phosphorylation was not detected at that time (Figure 3B, bottom panel); This was clearly observable only after 30 s of activation. Thus, it can be concluded that the change in the TCR that allows the recruitment of Nck precedes the phosphorylation of ITAMs in tyrosine. After analyzing the temperature dependence of the Nck-CD3ε junction and tyrosine phosphorylation, additional evidence was obtained in favor of complete independence of the Nck-CD3ε junction with respect to PTK activity. Total protein phosphorylation in activated tyrosine after TCR stimulation was decreased at 24 ° C and at 14 ° C and completely absent when cells were stimulated at 0 ° C (Figure 3C); while Nck binding to TCR was independent of temperature. Example 4.- TCR stimulation induces a conformational change that results in PRS exposure in CD3ε.

The fact that Nck-TCR binding is mediated by a SH3 domain suggests that binding of a ligand (antibody or antigen) to TCR promotes a conformational change that results in CD3ε PRS exposure. However, from previous experiments we cannot rule out possible contributions from other cellular factors. To eliminate this possibility, a cell-free assay was performed from extracts of Jurkat cells transfected with the Fv (single inoglobulin chain) fragment attached to the N-terminus of TCRβ. The Fv fragment that we used recognizes the NP and NIP haptens. By using columns of immobilized NP (low affinity hapten) and elution with free N1P (high affinity hapten), it was possible to purify and elute the entire TCR complex under mild conditions (data not shown). Once purified, the TCR was incubated with UCHT1 and precipitated with GST-SH3.1. Through this experiment we demonstrate that preincubation with anti-CD3 induces a conformational change in the purified TCR that allows its binding to the SH3.1 domain of Nck (Figure 4A). We have previously described that the APAl / 1 monoclonal antibody binds near the PRS region of CD3ε (Borroto et al., 1998). Incubation with APAl / 1 of purified TCR and pre-activated with UCHTl prevents its binding to GST-SH3.1 (Figure 4A). In contrast, APA1 / 2, a specific monoclonal antibody against the CD3δ cytoplasmic stem (Alarcon et al., 1991), had no effect.

To exclude that antibody crosslinking was the cause of the conformational change, a monovalent fragment of the anti-CD3 antibody was prepared and incubated with the purified TCR. As shown in Figure 4B, the Fab fragment induced the conformational change in the TCR resulting in exposure of the CD3ε site for Nck binding. A schematic of how ligand binding promotes both Nck recruitment and PTK activation is shown in Figure 4C.

Example 5.- The recruitment of Nck to the TCR is required for the activation of T lymphocytes.

The current model of signal transmission by the TCR places Nck well below the TCR. However, the characterization of the Nck-CD3ε interaction described above places Nck as an immediate effector of TCR. To understand the importance of Nck recruitment by the TCR in the activation of T lymphocytes we use two different approaches to inhibit the Nck-CD3ε interaction. In the first, we overexpressed the SH3.1 domain of Nck in order to occupy the PRS in CD3ε and prevent the interaction of TCR with endogenous Nck. Jurkat cells were stably transfected with a vector expressing a SH3.1-EGFP fusion protein and clones expressing the highest levels were selected by flow cytometry. Clones expressing EGFP alone or a fusion protein consisting of complete Nck bound to EGFP were also selected. It has been previously described that stimulation of Jurkat cells on anti-CD3 coated coverslips results in their extension and deployment in a few minutes, forming an F-actin ring that borders the periphery of the cells (Borroto et al., 2000; Bunnell et al., 2001). Since Nck is postulated to have a role in the rearrangement of the actin cytoskeleton (Buday, 1999), we performed a deployment test to test the effect of SH3.1 overexpression. Cells stably transfected with SH3.1-EGFP were less deployed and their actin cytoskeleton less polymerized than in cells expressing EGFP (Figure 5A). It is notable that although the level of expression of Nck-EGFP was lower than that of EGFP and SH3.1-EGFP (data not shown), there was a clear enhancement of cell deployment.

The unfolding of the cells takes place within a few minutes of stimulating the TCR. To examine the effect of SH3.1 overexpression on later processes, clones expressing SH3.1-EGFP, Nck-EGFP and EGFP were stimulated with plastic-bound anti-CD3 and the culture supernatant was collected 24 hr. then to analyze the release of IL-2. Similar to the effect on cell display, the Nck-EGFP-expressing clone produced more IL-2 after stimulating the TCR than the EGFP-expressing clone alone, whereas the SH3.1-expressing clone produced nothing (Figure 5B) . In contrast, Nck-EGFP and SH3.1-EGFP had no effect on CD69 expression (data not shown). To study cytokine release with a different stimulus, the clones were incubated with APCs preloaded with the SEE superantigen, and the release of IL-2 and TNFα to the culture supernatant was measured. SH3.1 expression again had a very strong inhibitory effect, reducing or even blocking SEE-induced release of both cytokines, whereas the complete Nck-transfected clone produced higher levels (Figure 5C and 5D).

The second approach consisted of transduction of T lymphocytes with the APA1 / 1 antibody, capable of blocking the Nck-CD3ε interaction by binding to PRS in CD3ε (Borroto et al, 1998). Peripheral blood mononuclear cells (PBMC) from healthy human donors were transduced with fluorescein-labeled APA 1/1. While anti-CD3 stimulation induced TCR to Nck association in non-transduced PBMC, the interaction was inhibited in APA1 / 1 transduced cells, suggesting that APA1 / 1 was blocking CD3ε PRS (Figure 5F). Finally, immobilized UCHT1 stimulated PBMC transduced either with APA1 / 1 or with a control antibody were stimulated and the effect on cell proliferation was analyzed 48 h later. We found that APAl / 1 internal expression inhibited TCR-induced proliferation at all antibody doses used (Figure 5E). Thus, blocking the site of interaction with Nck in CD3ε by overexpression of the SH3.1 domain of Nck or by transduction with a specific antibody, results in the inhibition of activation of T lymphocytes.

Example 6.- The overexpression of the Nck SH3.1 domain inhibits the formation of conjugates between T lymphocytes and APC and the maturation of the immune synapse. To investigate the mechanisms by which inhibition of Nck-interaction

CD3ε reduces the activation of T lymphocytes, we analyzed the formation of T: APC conjugates. TCR-induced signals increase integrin-mediated adhesion in a process dependent on the polymerization of the actin cytoskeleton (Dustin and Cooper, 2000). Since overexpression of the Nck SH3.1 domain inhibits TCR-stimulated induced actin polymerization, we predicted that conjugate formation would also be affected. This was confirmed in an experiment where Jurkat cells were stimulated with Raji APC loaded with SEE superantigen. Incubation with SEE produced a considerable increase in conjugate formation between Raji cells and the EGFP transfected Jurkat cell clone but not with the SH3.1 -EGFP transfected clone (Figure 6A).

Since TCR focuses on IS (Dustin and Cooper, 2000; Monks et al., 1998), we studied whether Nck's recruitment to TCR also results in his recruitment to IS. Using confocal videomicroscopy we found that Nck-EGFP concentrates on IS (Figure 6B) at a time similar to that of TCR recruitment (Krummel et al., 2000) and data not shown). In a similar experiment with cells expressing SH3.1-EGFP we were unable to see the translocation of this fusion protein to IS in the few cells that formed conjugates in the presence of SEE (data not shown). Anyway, We investigated the effect of SH3.1 overexpression on IS formation and maturation. Staining with anti-TCR antibodies showed that TCR accumulation in IS was not affected in SH3.1-EGFP-expressing cells compared to EGFP-expressing cells (Figure 6C), indicating that the Nck-CD3ε interaction is not essential for recruiting the TCR to the IS. Later we examined the association of PKCΘ with the SI. PKCΘ translocates to mature IS and is, in fact, an indicator of maturity (Dustin and Cooper, 2000; Monks et al, 1998). We found that PKCΘ translocation to IS was inhibited in cells expressing SH3.1-EGFP (Figure 6C). Making a quantification in this experiment, we found that the number of conjugates in which PKCΘ translocates to IS was reduced by three times (Figure 6D). Thus, the inhibition of TCR-mediated activation of T lymphocytes obtained by overexpression of the CD3ε binding Nck domain results from a double effect: a reduction in the number of T: APC conjugates formed and an inhibition of maturation of the IS.

Plasmids, cell lines, animals, antibodies and reagents are identified in the following Materials and Methods section, as well as a detailed description of the assays used in said Examples. MATERIALS AND METHODS

Cell Lines and Yeasts.- The human cell line of origin T named Jurkat was maintained in RPMI culture medium supplemented with fetal bovine serum (FBS, Sigma), 10%. The COS7 monkey cell line was maintained in DMEM medium supplemented with 5% FBS. The Jurkat line lacking the TCRβ 31.13 chain was kindly provided by Dr. A. Alcover (Institut Pasteur, Paris). The flag-CD3ε clone was obtained by stable transfection of the expression plasmid pSRα-FlagCD3ε in the Jurkat line. The clones named EGFP, Nck-EGFP and SH3.1-EGFP were obtained by stable transfection of the corresponding expression vectors in a Jurkat line named J77clon20 (Niedergang et al., 1997). The 31.13 scVβ3 clone was obtained by transfection of the expression vector for fusion of the Vβ3 chain fused with the single chain Fv. The Fv chain used is a derivative of the antibody against the hapten 3-nitro-4hydroxyphenylacetate Bl-8 (Reth et al., 1978). Thymus and spleen cells were isolated from transgenic mice with an AND-type TCR (Rag2 - / -) (Kaye et al., 1992) or from BL6 mice. As presenting cells they they used the DCEKs (Kuhlmam et al., 1991). The cd25h yeast strain was provided by Stratagene.

Plasmids.- The SOSε fusion protein was obtained by inserting the coding fragment by the cytoplasmic domain of human CD3ε into the yeast expression vector pSOS (Stratagene). The vector pSOSεtandem encodes the fusion of the SOS protein with two copies of the human tail-directed cytoplasmic domain of human CD3ε. Vectors encoding the control fusion proteins p SOS-MafB, pMyr-MafB, and pMyr-LamC were provided by Stratagene. For the GST-Nckβ and truncated GST-Nckβ fusion proteins, PCR fragments corresponding to the complete molecule or to a deletion of the amino terminal SH2 domain were obtained starting from the human Nckβ gene. These fragments were inserted into the vector pGEX4Tl in phase with the coding sequence for GST. The plasmids derived from pGEX4Tl, coding for the GST-Nckβ, GST-SH3.1α, GST-SH3.2α and GST-SH2α fusion proteins were kindly provided by Dr.R. Geha (Children's Hospital, Harvard Medical School, Boston ). The vectors encoding GST-SH2β and the HA epitope-tagged Nckα and Nckβ proteins were kindly assigned by Dr. X. Bustelo (National Cancer Center, Salamanca). The Ig-α immunoglobulin leader sequence, including the signal for peptidase cleavage and the Flag epitope, was amplified by PCR from the vector pEVmb-INneo (Schamel and Reth, 2000). The resulting fragment replaced the human leader sequence in the human CD3 chains in obtaining the expression vectors pSRαFlag-CD3ε, -CD3γ and -CD3δ. Cytoplasmic deletions of the human CD3ε chain were generated by a PCR-based mutagenesis method as previously described (Mallabiabarrena et al., 1992). The PCR products encoding the complete human Nckβ protein and its first SH3 domain were cloned into the mammalian expression vector pEFGPNl (Clontech), to obtain the Nck-EGFP and SH3.1-EGFP fusion proteins. Antibodies and Reagents.- The mouse antibodies APA1 / 1, SP34 and APA1 / 2, which recognize the cytoplasmic portion of human CD3ε, the extracellular portion of human CD3ε and the cytoplasmic tail of CD3δ respectively have been previously described (Alarcón et al. , 1991), as well as rabbit serum 448 specific against CD3ζ (San José et al., 2000) and mouse monoclonal antibody HP26 against human CD4 (Carrera y cois., 1987). The UCHTl mouse anti-CD3 monoclonal antibody was donated by Dr. P. Beverley (The Edward Jenner Institute for Vaccine Research, Berkshire, UK). The Fab fragments were prepared using the commercial Immunopure IgGl Fab Preparation Kit from Pierce. Mouse monoclonal antibody SP34 against the extracellular region of human CD3ε was donated by Dr. C. Terhost. The rabbit anti-Nck antibody, which recognizes both Nckα and Nckβ, was obtained from the Pharmingen house, the mouse anti-phosphotyrosine monoclonal antibody, 4G10, from Upstate Biotechnology, the mouse anti-HA monoclonal antibody, 12CA5, from Boehringer Manriheim and Sigma's anti-Flag mouse monoclonal antibody, M2. Staphylococcus aureus A and E enterotoxins, SEE and SEA, were purchased from Toxin Technologies. The Src family tyrosine kinase inhibitor, PP2, was purchased from Calbiochem. The TCR AND specific antigenic peptide, PCC, corresponding to amino acids 88-100 of pigeon cytochrome C, was synthesized by the method of N - (- 9-fluorenyl) methoxycarbonyl, (Fmoc), and purified by HPLC. All the culture media, amino acids and sugars necessary for the growth and transformation of the yeasts were purchased from Sigma and Merck.

Precipitation or "Pulí down" (Pd) Tests.- In the precipitation, or pulí down (Pd) tests, the different GST fusion proteins used throughout the present invention were always followed in the same way. Bacterial uses were prepared to absorb the fusion proteins to the glutathione sepharose matrix. The protocols followed for bacterial lysis and absorption to the matrix were those described by the commercial company of the GST system, Amersham Pharmacia Biotech. For each precipitation point, a volume of the matrix between 20 and 10 µl and a cell lysate of between 10 and 20 x 10 ⁶ cells were used. The Used were always pre-rinsed with the GST protein for 1 hour at 4 ° C under shaking conditions. Precipitation was then carried out under the same conditions for at least 4 hours or at most overnight. The precipitates were washed 3 to 5 times with the cell lysis buffer (Brij 96 0.3%, 150mM NaCl, 20mM Tris-HCl, pH 7.8, lOmM Iodoacetamide, lmM phenylmethylsulfonylfluoride, 1 µg / ml aproptinin, 1 µg / ml leupeptin, 1mM sodium orthovanadate and 20mM sodium fluoride). The precipitates were subjected to SDS-PAGE and transfer to nitrocellulose filters (BioRad). Screening of a cDNA library for ligands for the cytoplasmic domain of CD3ε using a two-hybrid system in yeast. - The system called Cytotrap by Stratagene was used. The yeast was cotransformed with the plasmid pSOS-CD3ε Tandem and the plasmid pMyr-library according to the protocols described by Stratagene. In the cotransformation 3 x 10 ⁶ cotransformants were analyzed, 15 clones were selected for their growth capacity under different temperature conditions and carbon source. The plasmid corresponding to the library of these 15 colonies was isolated and the growth capacity dependent on the cytoplasmic domain of CD3ε was analyzed by cotransforming these plasmids together with the empty plasmid pSOS. Through this procedure, two specific clones were selected that were sequenced to identify the corresponding proteins. Cell line transfections and metabolic labeling.- The transfection protocols of different cell types, Jurkat and COS7 lines, both transient and stable, used in the present invention have already been described (Borroto et al., 1998 and 1999; Pozo et al. ., 1999) The metabolic labeling of COS cells and Jurkat cells with ³⁵ S-Methionine (Amersham) has already been described (Borrroto et al., 1998). Stimulation and immunoprecipitation.- A total of between 20 and 10 x 10 ⁶ Jurkat cells per stimulation point are resuspended in 1 ml of 10mM RPMI HEPES medium, pH 7.4, not supplemented, and preincubated for 2 hours at 37 ° C before of stimulation. Then 10 µg of the stimulating antibody is added and the cells are incubated at 37 ° C during the stimulation time. Cells from stimulation were always used in Brij 96 0.3% lysis buffer. Pervanadate treatment performed in some cases for tyrosine kinase activation and phosphatase inhibition, in the absence of stimulus, has been previously described (Wienands et al., 1996). Treatment with the Src family tyrosine kinase inhibitor carried out during some stimulations consisted of pre-incubating the cells before stimulation for 30 minutes with the drug PP2 at a concentration of 20μM, which was maintained afterwards during stimulation. Stimulations of less than 1 minute in duration were performed differently. The cell suspension was concentrated by reducing its volume to half ml and was added already tempered at 37 ° C on an eppendorf with the 10 µg of stimulating antibody also tempered. On the other hand, the lysis buffer was prepared at double the final concentration of use and kept on ice in aliquots of half ml during the stimulation to immediately add the stimulated cells. After lysis, postnuclear fractions were obtained that were subjected to previously described immunoprecipitation protocols (Alarcón et al., 1991).

Purification of the TCR-CD3 complex.- The purification of the modified TCR-CD3 complex from the stable clone 31.13 was vβ3 with NP-sepharose and elution with NIP buffer was carried out as previously described (Schamel and Reth, 2000). Antibody transduction.- In order to introduce the APA1 / 1- Fitc and HP26-Fitc antibodies into living cells, the Gene Therapy Systems system called Bioporter Reagent was used. In-house specifications were followed, using the antibodies at a concentration of 250 µg / ml. The cells used were PBMCs isolated from healthy donor with Ficoll Hypaque (Rafer) and 2μl of the reagent was used for every 2 x 10 ⁶ cells. The efficiency of the process was monitored by flow cytometry, with 100% antibody-positive cells routinely observed. Functional Assays.- TCR stimulation-induced actin cytoskeleton polymerization was monitored by immunofluorescence. Cells were stimulated on coverslips treated with anti-CD3 antibodies (UCHTl) as already described (Borroto et al., 2000). Actin-F staining was performed by permeabilizing cells with 0.1% saponin and using phalloidin-Texas Red (Sigma). Images were analyzed with a Radiance 2000 confocal microscope. Cytokine secretion was quantified using an in-house system

Pharmingen called "Human Thl / Th2 Cytokine Cytometric Bead Assay kit". Jurkat cells were stimulated on wells of p96 culture plates, treated with the anti-CD3 antibody UCHTl for 24 hours, and the supernatants were analyzed with the kit. Alternatively the cells were stimulated with Raji presenter cells loaded 30 minutes previously with SEE in different doses, in a 1: 1 ratio.

To measure TCR-CD3 complex stimulation-induced proliferation, antibody-transduced PBMCs (1 x 10 ⁵ per dot) were seeded in p96 wells, treated with the anti-CD3 antibody UCHTl, at different doses, for 24 hours . Then 1 μCi of [ ³ H] thymidine (Amersham) per well was added and the incorporation of this metabolite was measured 24 hours later.

Conjugate formation and fluorescence in real time by confocal microscopy.- Raji cells were loaded with CMAC (10 mM for 20 minutes at 37 ° C in HBSS) and incubated for 20 minutes in the presence or absence of 5 µg / ml SEE. J77 clone 20 cells (5 x 10 ⁴ cells per well) were mixed with an equal number of Raji cells and plated onto poly-L-Lysine treated sheets within p24 plates (Costar). Cells were fixed and stained as previously described (Borroto et al., 1999, del Pozo et al., 1999). A quantitative analysis of the number of conjugates formed was performed by direct observation of the cell morphology and CMAC blue mark of the Raji cells with a fluorescence microscope (Leica DMR). The proportion of PKCΘ conjugates located in the contact region was calculated by randomly selecting 200 different conjugates. For real-time microscopy, J77 cells were placed in HBSS with FBS at

2% and absorbed onto fibronectin-treated coverslips (20 µg / ml for 20 hours at 4 ° C). The coverslips with the cells were mounted in an open chamber "Atto fluorine" (Molecular Probes). Then, CM-TMR loaded Raji cells (5mM for 20 min at 37 ° C) were added onto the chamber. Confocal images were acquired using a Leica TCS-SP confocal laser unit. Serial immunofluorescence and DIC images were obtained simultaneously at the indicated times. The most representative optical section of the green channel (EGFP, J77 cells), its corresponding DIC image (cell morphology) and the image of the red channel (CM-TMR, Raji cells) were superimposed to form a single image.

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Claims

1.- Modulating procedure for the activation of T lymphocytes characterized in that it is based on the regulation of the protein-protein interaction of CD3ε-Nck. 2. Method modulating the activation of T lymphocytes according to claim 1, characterized in that chemical substances are used that alter said interaction by their inhibition (antagonists). 3. Method modulating the activation of T lymphocytes according to claim 1, characterized in that chemical substances are used that alter said interaction through their activation (agonists). 4. Method modulating the activation of T lymphocytes according to any one of claims 2 and 3 characterized in that said agonist or antagonist substances are identified by a method comprising the following steps: a) generate a biological preparation of the protein-interaction protein that mimics the CD3ε-Nck interaction, b) add the candidate chemical substance to agonist or antagonist, and c) determine the blockage or activation of the protein-protein interaction CD3ε-Nck, identifying an antagonist of the CD3ε- interaction Nck and in case of activation an agonist of the CD3ε-Nck interaction. 5. Method modulating the activation of T lymphocytes according to claim 4 characterized in that the biological preparation of the protein-protein interaction of CD3ε- Nck is performed using the human CD3ε and Nck proteins, their homologous forms or peptides (fragments) that mimic the biological activity of said proteins or their homologous forms. 6. Method modulating the activation of T lymphocytes according to claim 4 or 5 characterized in that the peptide that mimics the biological activity of NcK binding to CD3ε comprises the amino acid sequence of the SH3.1 domain of NcKβ (SEQ ID NO2). 7. Method modulating the activation of T lymphocytes according to claim 4 or 5, characterized in that the peptide that mimics the biological activity of NcK binding to CD3ε comprises the amino acid sequence of the SH3.1 domain of NcKα (SEQ ID NO3). 8. Method modulating the activation of T lymphocytes according to claim 4 or 5, characterized in that the peptide that mimics the biological activity of Nck-binding CD3ε comprises the amino acid sequence of the proline-rich region (PRS) of CD3ε ( SEQ ID NO1). 9. Method modulating the activation of T lymphocytes according to any one of claims 4 to 8, characterized in that the amino acid sequence of human CD3ε and Nck proteins, their homologous forms or peptides (fragments) that mimic the biological activity of said proteins or their homologous forms is contained in a fusion protein. 10. Method modulating the activation of T lymphocytes according to claim 9, characterized in that the fusion protein is, among others, GST-SH3.1, SH3.1-EGFP, Nck-EGFP and HA-Nck. 11. Method modulating the activation of T lymphocytes according to any one of claims 4 to 10 characterized in that the human CD3ε and Nck proteins, their homologous forms or peptides (fragments) that mimic the biological activity of said proteins or their forms Homologues come from Cellular Used or from preparations of purified or semi-purified proteins. 12. Method modulating the activation of T lymphocytes according to any one of claims 4 to 11, characterized in that the biological preparation of the protein-protein interaction of CD3ε-Nck is carried out in a cell-free environment. 13. Method modulating the activation of T lymphocytes claim 12 characterized in that the cell-free environment is, among other possibilities, an immunoprecipitation test and a stimulation test of purified TCR. 14. Method modulating the activation of T lymphocytes according to any one of claims 4 to 13, characterized in that the protein-protein complexes that mimic the CD3ε-Nck interaction are identified, among other possibilities, by immunological techniques. 15. Method modulating the activation of T lymphocytes according to any one of claims 4 to 11 characterized in that the biological preparation of the protein-protein interaction of CD3ε-Nck is carried out in a cellular environment, among other possibilities, an assay Functional quantification of cell extension and deployment, a functional cytokine secretion quantification assay (IL-2 and TNFα), a functional assay for quantification of cell proliferation, a futional assay for quantification of the formation of conjugates between T lymphocytes and APC cells, a functional assay for quantification of IS maturation and a two-hybrid assay (trap assay). 16. Method modulating the activation of T lymphocytes according to any one of claims 4 to 15 characterized in that the biological preparation of the protein-protein interaction of CD3ε-Nck and the determination of blocking or activation of said interaction after the addition of an agonist or antagonist candidate is performed in a baseline or control situation, of stimulation (anti-CD3 and APC cells loaded with superantigen or antigen, among others) or blocking (overexpression of Nck SH3.1 and transduction of the APA1 antisense / 1, among others) of said interaction according to the interest in identifying one or another agent. 17. Method modulating the activation of T lymphocytes according to any one of claims 2 to 16 characterized in that the candidate chemicals to be tested as modulators of the CD3ε-Nck interaction can be produced, for example, by bacteria, yeasts and other microorganisms (natural products), chemically produced (for example, small molecules including peptidomimetics), or produced by genetic engineering. 18. Method modulating the activation of T lymphocytes according to claim 1 characterized in that it consists of a gene therapy treatment that allows the regulation of the protein-protein interaction of CD3ε and Nck. 19. Method modulating the activation of T lymphocytes according to claim 18, characterized in that the regulation is a blockade of the CD3ε-Nck interaction which is carried out, among other possibilities, by overexpression of the SH3.1 domain of Nck or by transduction with a specific anti-trust (APA1 / 1). 20.- Modulator procedure for the activation of T lymphocytes according to any one of claims 1-2 and 4 to 19 characterized in that diseases that are involved with a pathological activation of T lymphocytes are part of a treatment protocol such as , among others, autoimmune diseases such as rheumatoid arthritis, lupus erythematosus, multiple sclerosis, type I diabetes and myasthenia gravis; allergic diseases; in the prevention and treatment of transplant rejection and T lymphocyte lymphomas. 21. Modulation procedure for the activation of T lymphocytes according to any one of claims 1 and 3 to 18 characterized in that it is part of a protocol for the treatment of diseases that occur with an inhibition of T lymphocytes such as, among others, infectious, bacterial and viral diseases among others, and tumor. 22.- Sequence of nucleotides (DNA, RNA or cDNA) encoding human CD3ε and Nck proteins, their homologous forms or peptides (fragments) that mimic the biological activity of said proteins or their homologous forms characterized in that it is part of a process according to a any of claims 1 to 21. 23.- Expression vector characterized in that it contains a nucleotide sequence according to claim 22. 24.- Host cell characterized in that it contains an expression vector according to claim 23.