Method for generating t cells progenitors

Abstract

The invention relates to an in vitro method to generate T cell progenitors, comprising the step of culturing CD34+ cells in a medium containing TNF-alpha and/or an antagonist of the Aryl hydro-carbon/Dioxin receptor, in particular StemRegenin 1 (SR1), in presence of a Notch ligand and optionally a fibronectin fragment.

1 .- 20 . (canceled) 
     
     
         21 . An in vitro method for generating T cells precursors, comprising the step of culturing CD34+ cells in a medium comprising TNF-alpha and/or an antagonist of the Aryl hydrocarbon/Dioxin receptor, in particular StemRegenin 1 (SR1), and in the presence of an immobilized Notch ligand. 
     
     
         22 . The in vitro method of  claim 21 , wherein the Notch ligand is immobilized on the inner surface of a culture vessel or on the surface of beads present in the culture medium. 
     
     
         23 . The in vitro method of  claim 21 , wherein TNF-alpha and/or the antagonist of the Aryl hydrocarbon/Dioxin receptor is present, in the culture medium, from day 0 of the culture. 
     
     
         24 . The in vitro method of  claim 21 , wherein TNF-alpha is present, in the culture medium, at a concentration higher or equal to 3 ng/ml and/or wherein the antagonist of the Aryl hydrocarbon/Dioxin receptor is present, in the culture medium, at a concentration higher or equal to 30 ng/ml. 
     
     
         25 . The in vitro method of  claim 21 , wherein the CD34+ cells have been isolated from an adult donor. 
     
     
         26 . The in vitro method of  claim 21 , wherein the cells are cultured in the presence of TNF-alpha and/or SR1 for at most 10 days. 
     
     
         27 . The in vitro method of  claim 21 , wherein the cells are cultured in the presence of TNF-alpha and/or SR1 for between 3 and 7 days. 
     
     
         28 . The in vitro method of  claim 21 , wherein the Notch ligand is the soluble domain of the Delta-like-4 ligand, fused to an Fc region of an IgG protein. 
     
     
         29 . The in vitro method of  claim 21 , wherein the cells are also exposed to a fibronectin fragment, wherein said fragment comprises the RGDS and CS-1 patterns as well as a heparin-binding domain, preferably immobilized on the inner surface of the culture vessel or on beads. 
     
     
         30 . The in vitro method of  claim 21 , wherein the cells are also exposed to a fibronectin fragment, wherein the fibronectin fragment is RETRONECTIN®. 
     
     
         31 . The in vitro method of  claim 21 , wherein the culture medium also contains a vector intended for transfection or transduction of the CD34+ cells, during at least some time of exposure of the CD34+ cells to the Notch ligand. 
     
     
         32 . The in vitro method of  claim 31 , wherein the vector intended for transfection or transduction of the CD34+ cells is a transgene that codes for a Chimeric Antigen Receptor (CAR). 
     
     
         33 . The in vitro method of  claim 21 , further comprising the step(s) of:
 a. purifying the generated T cells progenitors, and   b. optionally conditioning the T cells progenitors in a pouch for injection into a patient.   
     
     
         34 . The in vitro method of  claim 21 , further comprising the step of:
 exposing the cells to a vector intended for transfection or transduction of CD34+ cells.   
     
     
         35 . The in vitro method of  claim 21 , further comprising the step of:
 exposing the cells to a vector or nucleic acid sequences containing the element appropriate for gene editing.   
     
     
         36 . A CD7+ T cell progenitors population wherein the proportion of cells that are CD34− and CD1a− or CD34− and CD5−, or CD34− and CD1a− and CD5− in this population is higher than 80%. 
     
     
         37 . The CD7+ T cell progenitors population according to  claim 36 , expressing a CAR. 
     
     
         38 . A method for increasing the number of T cells in a subject in need thereof, comprising administering progenitor T cells according to  claim 36  to a subject. 
     
     
         39 . The method of  claim 38 , for treating lymphopenia. 
     
     
         40 . The method of  claim 38 , for treating cancer, HIV infection, partial thymectomy, autoimmune disease, and/or organ transplant.