Cd4+cells with cytolytic properties

Abstract

The present invention relates to CD4+ T cells, more specifically cytolytic or cytotoxic CD4+ T-cells and methods of obtaining and identifying them.

1 . An isolated population of cytotoxic CD4+ T-cells characterised, when compared to natural CD4+ regulatory T-cells, by undetectable expression of the transcription repressor Foxp3. 
     
     
         2 . The population of cytotoxic CD4+ T-cells according to  claim 1 , further characterised, when compared to natural CD4+ regulatory T-cells, by an increased activity of the serine-threonine kinase AKT. 
     
     
         3 . The population of cytotoxic CD4+ T-cells according to  claim 1 , further characterised, when compared to natural CD4+ regulatory T-cells, by undetectable production of TGF-beta and undetectable or very low production of IL-10. 
     
     
         4 . The population of cytotoxic CD4+ T-cells according to  claim 1 , further characterised, when compared to natural CD4+ regulatory T-cells, by production of high concentrations of IFN-gamma. 
     
     
         5 . The population of cytotoxic CD4+ T-cells according to  claim 1 , fruther characterised, when compared to natural CD4+ regulatory T-cells, by production of high concentrations of soluble Fas ligand (FasL). 
     
     
         6 . An isolated population of cytotoxic CD4+ T-cells characterised, when compared to CD4+ effector cells, by co-expression of the transcription activators T-bet and GATA3 after antigenic stimulation. 
     
     
         7 . The population of cytotoxic CD4+ T-cells according to  claim 6  further characterised, when compared to CD4+ effector cells, by constitutive expression of cell surface proteins CD25 and GITR, and of intracellular CTLA-4. 
     
     
         8 . The population of cytotoxic CD4+ T-cells according to  claim 6 , further characterised, when compared to CD4+ effector cells, by expression of NKG2D. 
     
     
         9 . The population of cytotoxic CD4+ T-cells according to  claim 6 , or further characterised, when compared to CD4+ effector cells, by production of high concentrations of soluble Fas ligand (FasL). 
     
     
         10 . An isolated population of cytotoxic CD4+ T-cells characterised, when compared to NK cells, by expression of the CD4 co-receptor. 
     
     
         11 . The isolated population of cytotoxic CD4+ T-cells according to  claim 10  further characterised, when compared to Natural Killer cells (NK), by undetectable expression of CD49b. 
     
     
         12 . An isolated population of cytotoxic CD4+ T-cells characterised, when compared to Natural Killer T cells (NKT), by absence of expression of the invariant alpha chain of the T cell receptor. 
     
     
         13 . The isolated population of cytotoxic CD4+ T-cells according to  claim 12  further characterised, when compared to NKT cells, by expression of re-arranged beta chain of the T cell receptor. 
     
     
         14 . The isolated population of cytotoxic CD4+ T-cells according to  claim 12 , or further characterised, when compared to NKT cells, by lack of CD1d restriction. 
     
     
         15 . A method for obtaining in vivo or ex vivo the population of cytotoxic CD4+ T-cells according to  claim 1 , said method comprising the steps of:
 providing isolated natural naïve or memory CD4+ T-cells;   contacting said cells with an immunogenic peptide comprising a T-cell epitope and, adjacent to said T-cell epitope or separated therefrom by a linker of at most 7 amino acids, a C-(X)2-[CST] or [CST]-(X)2-C motif; and   expanding said cells in the presence of IL-2.   
     
     
         16 . A method of identifying a population of cytotoxic CD4+ T-cells, said method comprising the steps of:
 providing isolated natural CD4+ regulatory T-cells;   providing isolated CD4+ regulatory T-cells suspected of being cytotoxic; and   determining that the T-cells provided in (ii) display, compared to the T-cells provided in (i), undetectable expression of the transcription repressor Foxp3   
     
     
         17 . The method according to  claim 16 , said method further comprising determining in step (iii) that the T-cells provided in (ii) display, compared to the T-cells provided in (i), an increased activity of the serine-threonine kinase AKT. 
     
     
         18 . The method according to  claim 16 , said method further comprising determining in step (iii) that the T-cells provided in (ii) display, compared to the T-cells provided in (i), undetectable production of TGF-beta and undetectable or very low production of IL-10. 
     
     
         19 . The method according to  claim 16 , said method further comprising determining in step (iii) that the T-cells provided in (ii) display, compared to the T-cells provided in (i), production of high concentrations of IFN-gamma. 
     
     
         20 . The method according to  claim 16 , said method further comprising determining in step (iii) that the T-cells provided in (ii) display, compared to the T-cells provided in (i), production of high concentrations of soluble Fas ligand (FasL). 
     
     
         21 . A method of identifying a population of cytotoxic CD4+ T-cells, said method comprising the steps of:
 providing isolated natural CD4+ effector T-cells;   providing isolated CD4+ effector T-cells suspected of being cytotoxic; and   (iii) determining that the T-cells provided in (ii) display, compared to the T-cells provided in (i), co-expression of the transcription activators T-bet and GATA3.   
     
     
         22 . The method according to  claim 21 , said method further comprising determining in step (iii) that the T-cells provided in (ii) display, compared to the T-cells provided in (i) constitutive expression of cell surface proteins CD25 and GITR, and of intracellular CTLA-4. 
     
     
         23 . The method according to  claim 21 , said method further comprising determining in step (iii) that the T-cells provided in (ii) display, compared to the T-cells provided in (i) expression of NKG2D. 
     
     
         24 . The method according to  claim 21 , said method further comprising determining in step (iii) that the T-cells provided in (ii) display, compared to the T-cells provided in (i) high concentrations of soluble Fas ligand (FasL). 
     
     
         25 . A method of identifying a population of cytotoxic CD4+ T-cells, said method comprising the steps of:
 (i) providing isolated natural killer (NK) cells;   (ii) providing isolated CD4+ regulatory T-cells suspected of being cytotoxic; and   (iii) determining that the T-cells provided in (ii) display, compared to the T-cells provided in (i), the CD4 co-receptor.   
     
     
         26 . The method according to  claim 25 , said method further comprising determining in step (iii) that the T-cells provided in (ii) display, compared to the T-cells provided in (i), an undetectable expression of CD49b. 
     
     
         27 . A method of identifying a population of cytotoxic CD4+ T-cells, said method comprising the steps of:
 (i) providing isolated natural killer T (NKT) cells;   (ii) providing isolated CD4+ regulatory T-cells suspected of being cytotoxic; and   (iii) determining that the T-cells provided in (ii) display, compared to the T-cells provided in (i), absence of expression of the invariant alpha chain of the T cell receptor.   
     
     
         28 . The method according to  claim 27 , said method further comprising determining in step (iii) that the T-cells provided in (ii) display, compared to the T-cells provided in (i), expression of re-arranged beta chain of the T cell receptor. 
     
     
         29 . The method according to  claim 27 , said method further comprising determining in step (iii) that the T-cells provided in (ii) display, compared to the T-cells provided in (i), a lack of CD1d restriction.