Methods for modulating T cell responses by manipulating intracellular signal transduction

Abstract

Methods for modulating T cell responses by manipulating intracellular signals associated with T cell costimulation are disclosed. The methods involve inhibiting or stimulating the production of at least one D3-phosphoinositide in a T cell. Production of D3-phosphoinositides can be manipulated by contacting a T cell with an inhibitor or activator of phosphatidylinositol 3-kinase. Inhibitors of phosphatidylinositol 3-kinase for use in the methods of the invention include wortmannin and quercetin, or derivatives or analogues thereof. The methods of the invention can further comprise modulating other intracellular signals associated with costimulation, such as protein tyrosine phosphorylation, for example by modulating the activity of a protein tyrosine kinase or a protein tyrosine phosphatase in the T cell. Inhibition of a T cell response in accordance with the disclosed methods is useful therapeutically in situations where it is desirable to inhibit an immune response to an antigen(s), for example in organ or bone marrow transplantation and autoimmune diseases. Alternatively, stimulation of a T cell response in accordance with the disclosed methods is useful therapeutically to enhance an immune response to an antigen(s), for example to stimulate an anti-tumor response in a subject with a tumor, to stimulate a response against a pathogenic agent or increase the efficacy of vaccination. Novel screening assays for identifying inhibitors or activators of phosphatidylinositol 3-kinase, which can be used to inhibit or stimulate a T cell response, are also disclosed.

1 . A method for inhibiting a response by a T cell expressing a cell surface receptor which binds a costimulatory molecule, comprising contacting the T cell with an agent which inhibits production of D-3 phosphoinositides in the T cell.  
     
     
         2 . The method of  claim 1 , wherein the agent is an inhibitor of phosphatidylinositol 3-kinase.  
     
     
         3 . The method of  claim 2 , wherein the inhibitor of phosphatidylinositol 3-kinase is selected from a group consisting of wortmannin, quercetin and LY294002, and derivatives or analogues thereof.  
     
     
         4 . The method of  claim 1 , wherein the response by the T cell comprises production of at least one lymphokine.  
     
     
         5 . The method of  claim 4 , wherein the lymphokine is interleukin-2.  
     
     
         6 . The method of  claim 1 , wherein the response by the T cell comprises proliferation.  
     
     
         7 . The method of  claim 1 , further comprising contacting the T cell with a second agent which inhibits protein tyrosine phosphorylation in the T cell.  
     
     
         8 . The method of  claim 7 , wherein the second agent is an inhibitor of a protein tyrosine kinase.  
     
     
         9 . The method of  claim 8 , wherein the inhibitor of a protein tyrosine kinase is herbimycin A or a derivative or analogue thereof.  
     
     
         10 . The method of  claim 7 , wherein the second agent is a tyrosine phosphatase or an activator of a tyrosine phosphatase.  
     
     
         11 . The method of  claim 10 , wherein the tyrosine phosphatase is a cellular tyrosine phosphatase.  
     
     
         12 . The method of  claim 11 , wherein the cellular tyrosine phosphatase is CD45 or Hcph.  
     
     
         13 . The method of  claim 12 , wherein the second agent is a molecule which binds to and activates CD45.  
     
     
         14 . The method of  claim 13 , wherein the second agent is an anti-CD45 antibody, or fragment thereof.  
     
     
         15 . A method for inducing unresponsiveness to an antigen in a T cell expressing a cell surface receptor which binds a costimulatory molecule, comprising contacting the T cell with the antigen and an agent which inhibits production of D-3 phosphoinositides in the T cell.  
     
     
         16 . The method of  claim 15 , wherein the agent is an inhibitor of phosphatidylinositol 3-kinase.  
     
     
         17 . The method of  claim 16 , wherein the inhibitor of phosphatidylinositol 3-kinase is selected from a group consisting of wortmannin, quercetin and LY294002, and derivatives or analogues thereof.  
     
     
         18 . The method of  claim 15 , wherein the antigen is an alloantigen.  
     
     
         19 . The method of  claim 15 , wherein the antigen is an autoantigen.  
     
     
         20 . The method of  claim 15 , wherein the T cell is contacted with the antigen and the agent in vitro and the method further comprises administering the T cell to a subject.  
     
     
         21 . A method of  claim 20 , wherein the antigen is on a surface of an allogeneic or xenogeneic cell and the subject is a recipient of an allogeneic or xenogeneic cell.  
     
     
         22 . A method of  claim 20 , wherein the subject is suffering from an autoimmune disease or a disorder associated with an inappropriate or abnormal immune response.  
     
     
         23 . A method for stimulating a response by a T cell which has received a primary activation signal and expresses a surface receptor that binds a costimulatory molecule, comprising contacting the T cell with an agent which stimulates production of D-3 phosphoinositides in the T cell.  
     
     
         24 . The method of  claim 23 , wherein the agent is an activator of phosphatidylinositol 3-kinase.  
     
     
         25 . The method of  claim 23 , wherein the response by the T cell comprises production of at least one lymphokine.  
     
     
         26 . The method of  claim 25 , wherein the lymphokine is interleukin-2.  
     
     
         27 . The method of  claim 23 , wherein the response by the T cell comprises proliferation.  
     
     
         28 . The method of  claim 23 , further comprising contacting the T cell with a second agent which stimulates protein tyrosine phosphorylation in the T cell.  
     
     
         29 . The method of  claim 28 , wherein the second agent is an activator of a protein tyrosine kinase.  
     
     
         30 . The method of  claim 28 , wherein the second agent is an inhibitor of a cellular tyrosine phosphatase.  
     
     
         31 . The method of  claim 30 , wherein the cellular tyrosine phosphatase is CD45.  
     
     
         32 . A method for stimulating a response to an antigen by a T cell expressing a cell surface receptor which binds a costimulatory molecule comprising contacting the T cell with the antigen and an agent which stimulates production of D-3 phosphoinositides in the T cell.  
     
     
         33 . The method of  claim 32 , wherein the agent is an activator of phosphatidylinositol 3-kinase.  
     
     
         34 . The method of  claim 32 , wherein the antigen is a tumor-associated antigen.  
     
     
         35 . The method of  claim 32 , wherein the antigen is from a pathogen selected from the group consisting of a bacteria, a virus, a fungus and a parasite.  
     
     
         36 . The method of  claim 32 , wherein the T cell is contacted with the antigen and the agent in vitro and the method further comprises administering the T cell to a subject.  
     
     
         37 . A method of  claim 36 , wherein the antigen is expressed by a tumor cell present in the subject.  
     
     
         38 . A method of  claim 36 , wherein the antigen is expressed by a pathogen present in the subject.  
     
     
         39 . A method for identifying an inhibitor of a phosphatidylinositol 3-kinase comprising: 
 a) providing a T cell which expresses a receptor that binds a costimulatory molecule;    b) stimulating an intracellular signal transduction pathway in the T cell associated with ligation of the receptor in the presence of an agent to be tested; and    c) determining an amount of at least one D-3 phosphoinositide produced in the T cell,    wherein a reduced amount of at least one D-3 phosphoinositide produced in the T cell in the presence of the agent relative to an amount produced in the T cell in the absence of the agent indicates that the agent is an inhibitor of a phosphatidylinositol 3-kinase.    
     
     
         40 . The method of  claim 39 , wherein the receptor is CD28.  
     
     
         41 . The method of  claim 40 , wherein the T cell is contacted with a ligand for CD28.  
     
     
         42 . The method of  claim 40 , wherein the ligand for CD28 is a membrane-bound form of a B lymphocyte activation antigen selected from the group consisting of B7-1 and B7-2.  
     
     
         43 . The method of  claim 39 , wherein production of at least one D-3 phosphoinositide in the T cell is measured by high pressure liquid chromatography.  
     
     
         44 . A method for identifying an activator of phosphatidylinositol 3-kinase comprising: 
 a) contacting a T cell which expresses a receptor that binds a costimulatory molecule with an agent to be tested; and    b) determining an amount of at least one D-3 phosphoinositide produced in the T cell,    wherein an increased amount of at least one D-3 phosphoinositide produced in the T cell in the presence of the agent relative to an amount produced in the T cell in the absence of the agent indicates that the agent is an activator of a phosphatidylinositol 3-kinase.    
     
     
         45 . The method of  claim 44 , wherein production of at least one D-3 phosphoinositide in the T cell is measured by high pressure liquid chromatography.