Product and process to regulate actin polymerization in T lymphocytes

Abstract

The present invention relates to methods to regulate actin polymerization in T lymphocytes involved in tumorigenesis, inflammatory responses, immune responses, allergic responses and graft rejection responses, kits to perform such assays and methods to identify regulatory reagents that specifically control actin polymerization in T lymphocytes.

What is claimed is:  
     
         1 . A method to identify compounds capable of regulating actin polymerization in a T lymphocyte, comprising: 
 (a) contacting a putative regulatory compound with a T lymphocyte having a T cell receptor chain selected from the group consisting of a zeta chain and an epsilon chain, to form a contacted lymphocyte;    (b) combining said contacted lymphocyte with a molecule capable of inducing the phosphorylation of said zeta chain or said epsilon chain; and    (c) assessing the ability of said putative regulatory compound to regulate actin polymerization in said lymphocyte.    
     
     
         2 . The method of  claim 1 , further comprising assessing the amount of interleukin-2 produced by said lymphocyte.  
     
     
         3 . The method of  claim 1 , wherein said step of contacting comprises administering said putative regulatory compound by a technique selected from the group consisting of electroporation, microinjection, cellular expression, use of ion carrying agents and use of detergents for cell permeabilization.  
     
     
         4 . The method of  claim 3 , wherein said cellular expression is accomplished using an expression system selected from the group consisting of naked nucleic acid molecules, recombinant virus, retrovirus expression vectors and adenovirus expression vectors.  
     
     
         5 . The method of  claim 1 , wherein said method further comprises stimulating said lymphocyte, prior to said step of assessing, with a stimulatory compound selected from the group consisting of an antibody specific for a T cell receptor, an antibody specific for said ζ chain, an antibody specific for said ε chain, mitogens, lectins and major histocompatibility molecules associated with an antigenic peptide.  
     
     
         6 . The method of  claim 1 , wherein said method further comprises the step of determining when said lymphocyte has undergone a morphological change, said change selected from the group consisting of changes in adhesion, growth, shape and motility characteristics.  
     
     
         7 . The method of  claim 1 , wherein said method further comprises the step of determining when said lymphocyte has undergone a change in homing activity.  
     
     
         8 . The method of  claim 1 , further comprising determining the extent of actin association with said ζ chain or said ε chain in said lymphocyte by isolating said ζ or ε chain and detecting actin association using an antibody that specifically binds to actin.  
     
     
         9 . The method of  claim 1 , wherein said method further comprises determining the modulation of a molecule selected from the group consisting of focal adhesion kinase, paxillin, vinculin, tensin, PI-3Kα, TCR, Syk, Zap-70, Fyn, Shc, IRS-1, Nck, GRB-2, Lck, VAV, GAP, Raf, MEK, MEKK, MAPK, p38, JNKK, JNK, PLA2, JAK1, JAK2, JAK3, Tyk1, Tyk2, STATs, Myc, Jun, Ets-1, Elk-1, CREB, ATF-2, Yes, Hck, Src, CaM Kinase II, S6-K, sphingomyelinase, casein kinase, PKC, PI-3Kγ, SOS, CD45, HCP, Ssp, Syp, PLCγ1, PLCγ2, PLCβ1, PLCβ2, PLCβ3, PLCβ4, PLA2, Grb2, C5aR, IL-8R, MIP1αR, MIP1βR, MCP-1R, MCP-3R, PAFR, FMLPR, LTB 4 R, GRPR, and Fas receptor.  
     
     
         10 . The method of  claim 1 , wherein said method further comprises determining apoptosis of said lymphocytes.  
     
     
         11 . The method of  claim 1 , wherein said T lymphocyte is selected from the group consisting of T cell lines, T cell clones and T cell hybridomas.  
     
     
         12 . The method of  claim 1 , wherein said T cell receptor chain comprises at least one immunoreceptor tyrosine-based activation motif.  
     
     
         13 . The method of  claim 1 , wherein said T cell receptor chain comprises an immunoreceptor tyrosine-based activation motif selected from the group consisting of the amino acid sequence SEQ ID NO:1 and SEQ ID NO:2.  
     
     
         14 . A method to regulate actin polymerization in a T lymphocyte, comprising contacting a T lymphocyte with an effective amount of a regulatory reagent that is capable of altering the activity of an immunoreceptor tyrosine-based activation motif of a ζ chain of a T cell receptor.  
     
     
         15 . The method of  claim 14 , wherein said T lymphocyte is selected from the group consisting of a mature and an immature T lymphocyte.  
     
     
         16 . The method of  claim 14 , wherein said activity is altered by a mechanism selected from the group consisting of altering the interaction between said immunoreceptor tyrosine-based activation motif and its substrate, altering the interaction between said immunoreceptor tyrosine-based activation motif and its target molecule and altering the concentration of said immunoreceptor tyrosine-based activation motif in said lymphocyte.  
     
     
         17 . The method of  claim 14 , wherein said method regulates a T lymphocyte function selected from the group consisting of growth, differentiation, homing, proliferation, apoptosis and anergy.  
     
     
         18 . The method of  claim 14 , wherein said method regulates the production of interleukin-2.  
     
     
         19 . The method of  claim 14 , wherein said immunoreceptor tyrosine-based activation motif comprises the third immunoreceptor tyrosine-based activation motif of a T cell receptor zeta chain.  
     
     
         20 . The method of  claim 14 , wherein said immunoreceptor tyrosine-based activation motif comprises the amino acid sequence SEQ ID NO:1.  
     
     
         21 . The method of  claim 14 , wherein said regulatory reagent binds to said immunoreceptor tyrosine-based activation motif.  
     
     
         22 . The method of  claim 14 , wherein said regulatory reagent binds to an SH2 domain of a protein selected from the group consisting of a src-family kinase, a syk-family kinase and an adaptor molecule.  
     
     
         23 . The method of  claim 14 , wherein said regulatory reagent is selected from the group consisting of a protein-based compound, a carbohydrate-based compound, a lipid-based compound, a nucleic acid-based compound, a natural organic compound, a synthetically derived organic compound, an antibody and fragments thereof.  
     
     
         24 . The method of  claim 14 , wherein said regulatory reagent is selected from the group consisting of a peptide, a polypeptide and an antibody.  
     
     
         25 . The method of  claim 14 , wherein said regulatory reagent comprises an SH2 domain of a protein selected from the group consisting of Fyn, Lck, Zap-70, Shc, IRS-1, Nck, GRB-2, Syk, Yes, Hck, fak-B, PI-3 kinase and 14-3-3.  
     
     
         26 . The method of  claim 14 , wherein said regulatory reagent comprises an SH2 domain of a protein selected from the group consisting of Fyn, Lck, Zap-70, Shc, Syk, fak-B and 14-3-3.  
     
     
         27 . The method of  claim 14 , wherein said peptide is phosphorylated.  
     
     
         28 . The method of  claim 14 , wherein said regulatory reagent comprises an antibody specific for a protein selected from the group consisting of Fyn, Lck, Zap-70, Shc, IRS-1, Nck, GRB-2, Syk, Yes, Hck, fak-B, PI-3 kinase, 14-3-3, profilin, villin, fragmin, β-actin, gelsolin, depactin, focal adhesion kinase, paxillin, tensin, annexin, ezrin, clathrin-H chain, vinculin, talin, zixin, profilin, fractinin, cortactin, AFAP-110, p120; β catenin, connexin43 and cadherins.  
     
     
         29 . The method of  claim 14 , wherein said regulatory reagent is administered by at least one route selected from the group consisting of oral, nasal, topical, inhaled, transdermal, rectal, intraarticular and parenteral routes.  
     
     
         30 . The method of  claim 14 , wherein said regulatory reagent is administered by at least one route selected from the group consisting of topical, oral, aerosol, intravenous, intraarticular and intramuscular.  
     
     
         31 . The method of  claim 14 , wherein said method regulates T lymphocytes involved in a disease selected from the group consisting of an immunoproliferative disease, immunodeficiency disease, cancer, autoimmune disease, infectious disease, allergic response and graft rejection.  
     
     
         32 . The method of  claim 14 , wherein said effective amount reduces T cell receptor activation in said lymphocyte when compared with T cell receptor activation in lymphocytes that have not been contacted with said regulatory reagent.  
     
     
         33 . The method of  claim 14 , wherein said effective amount alters actin polymerization upon T cell receptor cross-linking as to actin polymerization resulting from T cell receptor cross-linking in the absence of said reagent.  
     
     
         34 . The method of  claim 14 , wherein said effective amount reduces production of interleukin-2 by said lymphocyte or induces the death of said lymphocyte.  
     
     
         35 . A method to regulate actin polymerization in a T lymphocyte, comprising contacting a T lymphocyte with an effective amount of a regulatory reagent that alters the activity of an immunoreceptor tyrosine-based activation motif of an ε chain of a T cell receptor.  
     
     
         36 . The method of  claim 35 , wherein said immunoreceptor tyrosine-based activation motif comprises the amino acid sequence SEQ ID NO:2.  
     
     
         37 . The method of  claim 35 , wherein said regulatory reagent binds to an SH2 domain of a protein selected from the group consisting of a src-family kinase, a syk-family kinase and an adaptor molecule.  
     
     
         38 . The method of  claim 35 , wherein said regulatory reagent comprises an SH2 domain of a protein selected from the group consisting of Fyn, Lck, Zap-70, Shc, IRS-1, Nck, GRB-2, Syk, Yes, Hck, fak-B, PI-3 kinase and 14-3-3.  
     
     
         39 . The method of  claim 35 , wherein said effective amount reduces production of interleukin-2 by said lymphocyte or induces the death of said lymphocyte.  
     
     
         40 . In a cellular system where a src-family tyrosine kinase is contacted with a T cell receptor chain selected from the group consisting of a ζ chain and an ε chain that is regulated by said src-family tyrosine kinase, the improvement comprising regulating actin polymerization by contacting a T lymphocyte with a reagent capable of binding to a protein selected from the group consisting of a third ITAM of a ζ chain, an ITAM of an ε chain and an SH2 domain.  
     
     
         41 . A formulation capable of regulating actin polymerization in a T lymphocyte, said formulation comprising: 
 (a) a regulatory reagent that alters the activity of a molecule selected from the group consisting of an immunoreceptor tyrosine-based activation motif of a ζ chain of a T cell receptor and an immunoreceptor tyrosine-based activation motif of a ε chain of a T cell receptor in a cell; and (b) a pharmaceutically acceptable carrier.    
     
     
         42 . The formulation of  claim 41 , wherein said formulation further comprises a compound selected from the group consisting of cytochalasins and a molecule that regulates a protein selected from the group consisting of focal adhesion kinase, paxillin, tensin, annexin, ezrin, clathrin-H chain, vinculin, talin, zixin, profilin, fractinin, cortactin, AFAP-110, p120, β catenin, connexin43 and cadherins.  
     
     
         43 . A kit to identify compounds capable of regulating actin polymerization in a T lymphocyte, said kit comprising: 
 (a) a cell comprising a T cell receptor chain selected from the group consisting of a ζ chain, an ε chain, and actin monomers; and    (b) a means for detecting the polymerization of said actin monomers.    
     
     
         44 . The kit of  claim 43 , wherein said cell is selected from the group consisting of Jurkat cells, HPB cells, MM-16.11 cells, MM-16.12 cells, MM-16.2 cells, MM-16.5 cells, 17.1 cells, MM-17.2 cells, 122.26 cells, HL-60 cells, H-9 cells, peripheral T cells, PBMC Cells, lymph node T cells, splenic T cells, thymocytes, intraepithelial lymphocytes and tumor infiltrating lymphocytes.  
     
     
         45 . The kit of  claim 43 , wherein said means for detecting comprises an antibody that binds specifically to actin.