Method for in vitro proliferation of dendritic cell precursors and their use to produce immunogens

Abstract

A method for producing proliferating cultures of dendritic cell precursors is provided. Also provided is a method for producing mature dendritic cells in culture from the proliferating dendritic cell precursors. The cultures of mature dendritic cells provide an effective means of producing novel T cell dependent antigens comprised of dendritic cell modified antigens or dendritic cells pulsed with antigen, including particulates, which antigen is processed and expressed on the antigen-activated dendritic cell. The novel antigens of the invention may be used as immunogens for vaccines or for the treatment of disease. These antigens may also be used to treat autoimmune diseases such as juvenile diabetes and multiple sclerosis.

We claim:  
     
         1 . A method of producing a population of dendritic cell precursors from proliferating cell cultures, comprising 
 a) providing a tissue source comprising dendritic cell precursors;    b) culturing the tissue source on a substrate in culture medium comprising GM-CSF and another factor which increases the proportion of dendritic cell precursors by inhibiting the proliferation or maturation of non-dendritic cell precursors to produce proliferating dendritic cell precursors; and    c) culturing the dendritic cell precursors for a period of time sufficient to allow them to mature into mature dendritic cells.    
     
     
         2 . The method of  claim 1 , wherein said agent inhibits macrophage proliferation or maturation.  
     
     
         3 . The method of  claim 1 , wherein said tissue source is human blood.  
     
     
         4 . The method of  claim 3 , wherein said agent is selected from the group consisting of IL-4 and IL-13.  
     
     
         5 . The method of  claim 4 , wherein said agent is IL-4.  
     
     
         6 . The method of  claim 5 , wherein IL-4 is present in the culture medium in the range of 500-1000 U/ml.  
     
     
         7 . The method of  claim 1 , wherein the culture medium further comprise TNF-α.  
     
     
         8 . The method according to  claim 1  wherein fetal calf serum is present in the culture medium in an amount of about 1 to 15%.  
     
     
         9 . The method according to  claim 1  wherein the fetal calf serum is present in the culture medium in an amount of about 10%.  
     
     
         10 . The method according to  claim 3  wherein GM-CSF is present in the medium at a concentration of about 1-1000 U/ml.  
     
     
         11 . The method according to  claim 10  where the GM-CSF is present in the medium at a concentration of about 400-800 U/ml.  
     
     
         12 . The method according to  claim 11 , wherein the GM-CSF is present at a concentration of about 800 U/ml.  
     
     
         13 . The method according to  claim 1  wherein at least one factor selected from the group consisting of TNF-α, G-CSF, IL-1 and IL-3 is present in the culture medium.  
     
     
         14 . A composition comprising a dendritic cell modified antigen wherein a substance to be modified is exposed to a culture of dendritic cells prepared according to  claim 1  and whereby the substance is modified by the dendritic cells to produce the modified antigen.  
     
     
         15 . A method of immunizing against disease in humans or animals comprising, administering an immunogenic amount of the composition of  claim 14 .  
     
     
         16 . A vaccine comprising the composition of  claim 14 .  
     
     
         17 . A composition comprising antigen activated dendritic cells wherein dendritic cells prepared according to  claim 1  are pulsed with an antigen and wherein the dentritic cells process the antigens to produce a modified antigen which is expressed by dendritic cells.  
     
     
         18 . A method of treating autoimmune disease comprising administering to a person in need of treatment a therapeutically effective amount of the composition of  claim 14  and wherein the antigen to be modified is a self-protein.  
     
     
         19 . A method of treating autoimmune disease comprising administering to a person in need of treatment a therapeutically effective amount of the composition of  claim 17  wherein the antigen to be modified is a self-protein.  
     
     
         20 . The method of claims  18  or  19  wherein the autoimmune disease is selected from the group consisting of multiple sclerosis myasthenia gravis, atopic dermatitis and juvenile diabetes.  
     
     
         21 . The method of claims  18  or  19  wherein the autoimmune disease is selected from the group consisting of multiple sclerosis and juvenile diabetes.  
     
     
         22 . Dendritic cell precursors prepared according to the method of  claim 1 .  
     
     
         23 . The method according to  claim 1  wherein cord blood serum is present in the culture medium in an amount of about 5%.