Detection and quality control of regulatory T cells through DNA-methylation analysis of the Foxp3 gene
The present invention relates to a method, in particular an in vitro method for identifying FoxP3-positive regulatory T cells, preferably CD25 + CD4 + regulatory T cells of a mammal, comprising analysing the methylation status of at least one CpG position in the gene foxp3 or an orthologous or paralogous gene thereof, and the use of 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.
1. A method of identifying stable FoxP3 positive regulatory T cells and treating a disease in a human subject, the method comprising:
a) obtaining a sample containing human regulatory T cells from a donor or a cell culture;
b) identifying stable FoxP3 positive human regulatory T cells from a portion of the sample comprising:
i) isolating genomic DNA from the portion of the sample;
ii) treating the isolated genomic DNA with bisulfite to convert unmethylated cytosines to uracils;
iii) amplifying the treated isolated genomic DNA by polymerase chain reaction (PCR) using a pair of oligonucleotide primers comprising SEQ ID NO: 13 and SEQ ID NO: 14, or SEQ ID NO: 15 and SEQ ID NO: 16, or SEQ ID NO: 17 and SEQ ID NO: 18, or SEQ ID NO: 29 and SEQ ID NO: 30 to produce an amplicon;
iv) sequencing the amplicon; and
v) analyzing the amplicon for a lack of methylation at cytosine phosphate guanine (CpG) positions in the human FoxP3 gene in the amplicon, wherein a lack of methylation at the CpG positions is indicative of stable FoxP3 positive human regulatory T cells in the sample; and
c) administering a portion of the sample from step a), wherein the sample contains stable FoxP3 positive human regulatory T cells as identified in step b) to thereby treat an autoimmune disease, adverse effects in allotransplant recipients, tumorous diseases, allergic asthma, ovarian cancer, chronic graft-versus host disease, multiple sclerosis, or immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome in the human subject.
2. The method according to claim 1 , further comprising inducing the cells in said sample with transforming growth factor-β (TGF-β).
3. The method according to claim 1 , further comprising identifying said FoxP3-positive human regulatory T cell as a CD25+CD4+ T cell.
4. The method according to claim 1 , wherein said oligonucleotide primers SEQ ID NO:13 and SEQ ID NO:14 are used to amplify said treated genomic DNA.
5. The method according to claim 1 , wherein said oligonucleotide primers SEQ ID NO:15 and SEQ ID NO:16 are used to amplify said treated genomic DNA.
6. The method according to claim 1 , wherein said oligonucleotide primers SEQ ID NO:17 and SEQ ID NO:18 are used to amplify said treated genomic DNA.
7. The method according to claim 1 , comprising amplifying the treated isolated genomic DNA by PCR using a pair of oligonucleotide primers comprising.
8. The method according to claim 1 , wherein the portion of the sample from step a) administered to the human subject is further identified as CD25+CD4+ T cells.
9. The method according to claim 1 , wherein the pair of oligonucleotide primers consist of SEQ ID NO: 13 and SEQ ID NO: 14.
10. The method according to claim 1 , wherein the pair of oligonucleotide primers consist of SEQ ID NO: 15 and SEQ ID NO: 16.
11. The method according to claim 1 , wherein the pair of oligonucleotide primers consist of SEQ ID NO: 17 and SEQ ID NO: 18.
12. The method according to claim 1 , wherein the pair of oligonucleotide primers consist of SEQ ID NO:29 and SEQ ID NO:30.