Epigenetic Markers for the Identification of Blood Sub-cells of Type 1

Abstract

The present invention relates to a method, in particular an in vitro method, for identifying CD3CD4 positive T lymphocytes of a mammal, wherein said method comprises analysing the methylation status of at least one CpG position in the CD3a/b/c/d/g genes, in particular their “upstream” regulatory regions, and in particular the promoter and other conserved regions of the gene cd3, wherein a demethylation of at least one CpG in the analyzed sample to at least 90% is indicative for memory and naive CD4 or/and memory and/or naïve T lymphocytes. Furthermore, the present invention is directed at the use of DNA-methylation analysis of the genes CD3a/b/c/d for the detection and quality assurance and control of T lymphocytes. Furthermore, the present invention relates to a kit for performing the above methods as well as respective uses thereof. In a preferred embodiment, the present invention furthermore provides an improved method for analysing the methylation status of at least one CpG position in the gene CD3, allowing for a precise analysis even from sub-optimal quality samples, such as non-freshly obtained blood or serum samples.

We claim: 
     
         1 . A method for identifying CD3 +  CD4 +  and/or CD3 +  CD8 +  naïve and/or memory T-lymphocytes in a sample derived from a human comprising T cells, the method comprising:
 a) obtaining a sample comprising T cells from a human, 
 b) isolating DNA from said T cells comprising a sequence according to SEQ ID NO: 1, and treating said DNA with bisulfite, 
 c) amplifying an amplicon from said treated DNA using bisulfite-specific primer pairs according to SEQ ID NOs: 70 and 71 and SEQ NOs: 72 and 73, 
 d) analyzing the methylation status of at least one CpG position in said amplicon as amplified in step c), and 
 e) identifying CD3 +  CD4 +  and/or CD3 +  CD8 +  naïve and/or memory T-lymphocytes based on said methylation status, wherein a demethylation of at least one CpG position in said amplicon to at least 90% is indicative for a CD3 +  CD4 +  and/or CD3 +  CD8 +  naïve and/or memory T-lymphocyte cell. 
 
     
     
         2 . The method according to  claim 1 , wherein said at least one CpG position is demethylated to more than 91%. 
     
     
         3 . The method according to  claim 1 , further comprising the step of discriminating said CD3 +  CD4 +  and/or CD3 +  CD8 +  naïve and/or memory T-lymphocytes from other leukocytes in said sample based on said methylation status, wherein a demethylation of at least one CpG position in said amplicon to at least 90% is indicative for a CD3 +  CD4 +  and/or CD3 +  CD8 +  naïve and/or memory T-lymphocyte cell. 
     
     
         4 . The method according to  claim 1 , wherein said analysis of the methylation status further comprises a method selected from bisulfite sequencing, MSP, HeavyMethyl, MethyLight, and Ms-SNuPE. 
     
     
         5 . The method according to  claim 1 , wherein said method is suitable for application on a DNA-chip. 
     
     
         6 . The method according to  claim 1 , wherein said identification comprises a distinction of said T-lymphocytes from all major peripheral blood cell types or non-blood cells. 
     
     
         7 . The method according to  claim 1 , wherein said human suffers from or is likely to suffer from an autoimmune disease, a transplant rejection, cancer, and/or an allergy. 
     
     
         8 . The method according to  claim 1 , the method further comprising the steps of:
 f) quantifying the amount of CD3 +  CD4 +  and/or CD3 +  CD8 +  naïve and/or memory T-lymphocytes in said sample from step a) based on said identification in step e),   g) quantifying the amount of T-lymphocytes in an earlier sample from said human or in a control sample comprising an identification according to steps b) to e), and   h) comparing the amount of T-lymphocytes in the present sample from step (a) to said earlier sample or to the control sample.   
     
     
         9 . The method according to  claim 8 , wherein said human suffers from or is likely to suffer from an autoimmune disease, a transplant rejection, cancer, and/or an allergy. 
     
     
         10 . The method according to  claim 1 , wherein said method further comprises analysing the amplicon to determine if it has sequences according to SEQ ID NOs: 6, 7, or 8. 
     
     
         11 . A method for quantifying the methylation of at least one CpG position in a sequence according to SEQ ID NO: 1, the method comprising:
 a) obtaining a sample comprising T cells from a human,   b) isolating DNA from said T cells comprising a sequence according to SEQ ID NO: 1, and treating said DNA with bisulfite,   c) amplifying an amplicon from said treated DNA using bisulfite-specific primer pairs according to SEQ ID NOs: 70 and 71 and SEQ ID NOs: 72 and 73,   d) quantifying the methylation of the at least one CpG position in said amplicon as amplified in step c),   wherein a demethylation of the least one CpG position in said amplicon to at least 90% is indicative for a CD3 +  CD4 +  and/or CD3 +  CD8 +  naïve and/or memory T-lymphocyte cell.   
     
     
         12 . The method, according to  claim 11 , wherein step d) comprises quantifying the methylation of a combination of at least two CpG positions in said amplicon as amplified in step c),
 wherein a demethylation of a combination of at least two CpG positions in said amplicon to at least 90% is indicative for a CD3 +  CD4 +  and/or CD3 +  CD8 +  naïve and/or memory T-lymphocyte cell.   
     
     
         13 . A method for identifying CD3 +  CD4 +  and/or CD3 +  CD8 +  naïve and/or memory T-lymphocytes in a sample derived from a human comprising T cells, the method comprising:
 a) obtaining a sample comprising T cells from a human, 
 b) isolating DNA from said T cells comprising a sequence according to SEQ ID NO: 1, and treating said DNA with bisulfite, 
 c) performing quantitative Polymerase Chain Reaction on the bisulfite-treated DNA using bisulfite-specific primer pairs and a bisulfite-specific probe, 
 d) analyzing the methylation status of at least one CpG position in said amplicon as amplified in step c), and 
 e) identifying CD3 +  CD4 +  and/or CD3 +  CD8 +  naïve and/or memory T-lymphocytes based on said methylation status, wherein a demethylation of at least one CpG position in said amplicon to at least 90% is indicative for a CD3 +  CD4 +  and/or CD3 +  CD8 +  nave and/or memory T-lymphocyte cell. 
 
     
     
         14 . The method, according to  claim 13 , wherein the bisulfite-specific primer pair is SEQ ID NOs: 76 and 77 and the bisulfite-specific probe is SEQ ID NO: 78. 
     
     
         15 . The method, according to  claim 13 , wherein the bisulfite-specific primer pair is SEQ ID NO: 79 and 80 and the bisulfite-specific probe is SEQ ID NO: 81. 
     
     
         16 . An oligomer having to any of SEQ ID NOs: 70 to 73, 76, 77, 79, and 80 or an amplicon having SEQ ID NO: 6 to 8. 
     
     
         17 . A kit for identifying and/or monitoring CD3 +  T-lymphocytes, in particular CD3 +  CD4 + , or CD3 +  CD8 +  T-lymphocytes, in a mammal based on the analysis of the methylation status of CpG positions in the gene CD3, comprising materials for performing a method according to  claim 1 . 
     
     
         18 . The kit according to  claim 17 , comprising a) a bisulfite reagent, and b) materials for the methylation analysis of CpG positions selected from the positions consisting of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 of an amplicon according to SEQ ID NO: 6, 7, or 8.