Method for obtaining antigen-specific tr1 regulatory lymphocytes

Abstract

The invention relates to a method for preparing antigen-specific Tr1 regulatory lymphocytes. The inventive method involves the use of artificial antigen-presenting cells, expressing a molecule from the HLA class II system and a human LFA-3 molecule and expressing none of the B7-1, B7-2, B7-H1, CD40, CD23 or ICAM-1 costimulation molecules.

1 . A method of treating an inflammatory disease and/or autoimmune disease in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of antigen-specific Tr1 cells obtained from activation and differentiation of CD4+ lymphocytes of a blood or serum sample of said patient. 
     
     
         2 . The method according to  claim 1 , wherein said inflammatory disease and/or autoimmune diseases is Crohn's disease. 
     
     
         3 . The method according to  claim 1 , wherein said inflammatory disease and/or autoimmune diseases is multiple sclerosis. 
     
     
         4 . The method according to  claim 1 , wherein said inflammatory disease and/or autoimmune diseases is diabetes. 
     
     
         5 . The method according to  claim 1 , wherein said inflammatory disease and/or autoimmune diseases is atherosclerosis. 
     
     
         6 . The method according to  claim 1 , wherein said inflammatory disease and/or autoimmune diseases is rheumatoid polyarthritis. 
     
     
         7 . The method according to  claim 1 , wherein said inflammatory disease and/or autoimmune diseases is psoriasis. 
     
     
         8 . The method according to  claim 1 , wherein said inflammatory disease and/or autoimmune diseases is asthma. 
     
     
         9 . The method according to  claim 1 , wherein said antigen-specific Tr1 cells are obtained by:
 separating lymphocytes from a sample of a subject's blood or serum,   activating said lymphocytes in vitro by co-culturing with artificial antigen-presenting cells comprising cells which express a class II molecule of the HLA system and a human LFA-3 molecule and which do not express any of the costimulatory molecules B7-1, B7-2, B7-Hl, CD40, CD23 or ICAM-1, and having a selected antigen associated with them,   recovering from said activated lymphocytes a population of activated CD4+ T lymphocytes comprising at least 10% of Tr1 lymphocytes which are specific for the antigen of the antigen-presenting cells.   
     
     
         10 . The method according to  claim 9 , wherein said artificial antigen-presenting cells are obtained by co-transfection of mammalian cells selected from the group consisting of fibroblasts, keratinocytes, kidney tubule cells, Schwann cells, myoblasts and endothelial cells, with a nucleic acid sequence the α chain of a class II HLA molecule, a nucleic acid sequence encoding the β chain of a class II HLA molecule and a nucleic acid sequence encoding human LFA-3. 
     
     
         11 . The method according to  claim 1 , wherein said antigen-specific Tr1 cells are obtained by:
 separating lymphocytes from a sample of a subject's blood or serum,   activating said lymphocytes in vitro by co-culturing with artificial antigen-presenting cells comprising cells which express a class II molecule of the HLA system and a human LFA-3 molecule and which do not express any of the costimulatory molecules B7-1, B7-2, B7-H1, CD40, CD23 or ICAM-1, and having a selected antigen associated with them,   repeating the steps in which CD4+ T lymphocytes are activated in vitro with the selected antigen and the antigen-presenting cells, and   separating the desired Tr1 lymphocyte population when the population has been enriched to at least 30% Tr1 lymphocytes that are specific for the selected antigen.   
     
     
         12 . The method according to  claim 1 , wherein said antigen-specific Tr1 cells are obtained by:
 separating lymphocytes from a sample of a subject's blood or serum,   activating said lymphocytes in vitro by co-culturing with artificial antigen-presenting cells comprising cells which express a class II molecule of the HLA system and a human LFA-3 molecule and which do not express any of the costimulatory molecules B7-1, B7-2, B7-H1, CD40, CD23 or ICAM-1, and having a selected antigen associated with them,   repeating the steps in which CD4+ T lymphocytes are activated in vitro with the selected antigen and the antigen-presenting cells, and   separating the desired Tr1 lymphocyte population when the population has been enriched to at least 30% Tr1 lymphocytes that are specific for the selected antigen   isolating Tr1 lymphocyte clones from the recovered enriched cell population; and   propagating the isolated Tr1 lymphocyte clones.