Method for the detection, preparation and depletion of cd4+ t lymphocytes

Abstract

The present invention relates an in vitro method for detecting class II restricted CD4+ T cells in a sample. Herein a sample is contacted with an isolated complex of an MHC class II molecule and a peptide. This peptide comprises an MHC class II restricted T cell epitope of an antigenic protein and immediately adjacent thereof, or separated by a linker of at most 7 amino acids a sequence with a [CST]-xx-C or C-xx-[CST] motif. CD4+ T cells are detected by measuring the binding of the complex with cells in the sample, wherein the binding of the complex to a cell is indicative for the presence of CD4+ T cells in the sample. The present invention further relates to an isolated complex of an MHC Class II molecule and a peptide comprising an MHC class II restricted T cell epitope of an antigenic protein and immediately adjacent thereof, or separated by a linker of at most 7 amino acids a sequence with a [CST]-xx-C or C-xx-[CST] motif.

1 - 30 . (canceled) 
     
     
         31 . An in vitro method for detecting class II restricted CD4+ T cells in a sample comprising the steps of:
 providing a sample,   contacting the sample with a complex of an isolated MHC class II molecule and a peptide, said peptide comprising an MHC class II restricted T cell epitope of an antigenic protein and immediately adjacent thereof, or separated by a linker of at most 7 amino acids, a sequence with a [CST]-X(2)-C or C—X(2)-[CST] redox motif, wherein X is any amino acid,   measuring the binding of said complex with cells in said sample, wherein the binding of said complex to a cell is indicative for the presence of CD4+ T cells in said sample.   
     
     
         32 . The method according to  claim 31 , wherein said redox motif is C—X(2)-C. 
     
     
         33 . The method according to  claim 31 , wherein said linker has a length of maximum 4 amino acids. 
     
     
         34 . The method according to  claim 31 , wherein said peptide occurs in a non-covalent complex with the MHC class II molecule and has a length of between 12 and 20 amino acids. 
     
     
         35 . The method according to  claim 31 , wherein said complex is a fusion protein of said peptide and an MCH class II molecule. 
     
     
         36 . The method according to  claim 31 , wherein the sample is a blood sample or a tissue sample. 
     
     
         37 . The method according to  claim 36 , wherein the tissue sample is synovial fluid from rheumatoid arthritis patients or pleural fluid of patients with infectious pneumonia. 
     
     
         38 . The method according to  claim 31 , wherein said CD4+ in a sample cells are one or more selected form the group consisting of Tregs, induced Tregs such as Tr1 or Th3, naïve CD4+ T cells, polarised CD4+ T cells and cytolytic CD4+ T cells. 
     
     
         39 . The method according to  claim 31 , wherein said sample comprises one or more of the group consisting of naïve CD4+ T cells, antigen-exposed CD4+ T cells, CD4+ T cells obtained during therapy or during vaccination and CD4+ T cells in a tissue. 
     
     
         40 . The method according to  claim 31 , wherein said epitope sequence of said peptide is identical to the epitope sequence of said antigenic protein or wherein, the MHC class II anchoring residues sad the epitope sequence of said peptide are modified compared to the epitope sequence of said antigenic protein in order to modulate the binding affinity of an epitope to CD4+ cells. 
     
     
         41 . The method according to  claim 31 , wherein said MHC class II molecules in the complex are multimers. 
     
     
         42 . The method according to  claim 31 , wherein said complex is a soluble complex. 
     
     
         43 . The method according to  claim 36 , wherein said complex is attached to an insoluble carrier or a substrate. 
     
     
         44 . The method according to  claim 31 , further comprising the step of isolating CD4+ T cells which are bound to said complex. 
     
     
         45 . The method according to  claim 36 , further comprising the step of detecting or isolating subpopulations of detected CD4+ T cells. 
     
     
         46 . An isolated complex of:
 An MCH class II molecule and   a peptide comprising an MHC class II restricted T cell epitope of an antigenic protein and immediately adjacent thereof, or separated by a linker of at most 7 amino acids a sequence with a [CST]-X(2)-C or C—X(2)-[CST] redox motif, wherein X is any amino acid.   
     
     
         47 . The complex according to  claim 46 , wherein said redox motif is C—X(2)-C. 
     
     
         48 . The complex according to  claim 46 , wherein said linker has a length of maximum 4 amino acids. 
     
     
         49 . The complex according to  claim 46 , wherein said complex between said peptide and said MHC class II molecule is a covalently bound complex. 
     
     
         50 . The complex according to  claim 49 , wherein said covalently bound complex is a fusion protein of an MHC class II protein with said peptide. 
     
     
         51 . The complex according to  claim 46 , wherein said complex is attached to a bead or a plate.