DNA methylation analysis of regulatory T cells through DNA-methylation analysis of the TSDR region of the gene FOXP3

摘要

The present invention relates to a method, in particular an in vitro method for identifying FoxP3-positive CD25+CD4+ regulatory T cells of a mammal, comprising analyzing the methylation status of at least one CpG position in the FOXP3 gene, in particular its “upstream” regulatory regions, and in particular the promoter and the TSDR region of the gene foxp3, wherein a demethylation to at least 90% of at least one CpG in the sample as analyzed is indicative for a FoxP3-positive CD25+CD4+ regulatory T cell, and the use of said DNA-methylation analysis of the gene of the transcription factor FoxP3 for a detection and quality assurance and control of regulatory T cells. Furthermore, the present invention relates to a kit for performing the above methods as well as respective uses. The present invention furthermore provides an improved method for analyzing the methylation status of at least one CpG position in the gene foxp3 that allows for a precise analysis even from sub-optimal quality samples, such as non-freshly obtained blood or serum samples.

主权项

1. A method for identifying FoxP3-positive CD25+CD4+ regulatory T cells (Tregs) of a human subject, the method comprising:
Isolating genomic DNA from a biological sample from the subject, and treating the genomic DNA with bisulfite, detecting, in the bisulfite treated genomic DNA, the methylation status of at least one CpG position in the region of the foxp3 gene consisting of SEQ ID NO: 1; wherein said detecting the methylation status comprises creating an amplicon by amplification with at least one methylation-specific primer having the sequence of SEQ ID NO: 14 or 16, and at least one non-methylation-specific primer having the sequence of SEQ ID NO: 15 or 17, and hybridizing the resulting amplicon with at least one suitable methylation-specific probe and/or at least one suitable non-methylation-specific probe; determining that said at least one CpG position is demethylated to at least 90%: and correlating the detected extent of demethylation of said at least one CpG position with an identification the sample as having FoxP3-positive CD25+CD4+ regulatory T cells.