Epigenetic method for the identification of follicular T-helper-(TFH-) cells
The present invention relates to a method, in particular an in vitro method, for identifying follicular helper T cells, comprising analyzing the methylation status of at least one CpG position in the mammalian gene region for leukemia inhibitory factor (LIF), wherein a demethylation of said gene region is indicative for a follicular helper T cell, when compared to a non-follicular helper T cell. The analyses according to the invention can identify follicular helper T cells on an epigenetic level and distinguish them from all other cells in complex samples, such as, for example, other blood cells. The present invention furthermore provides an improved method for quantifying follicular helper T cells in complex samples. The method can be performed without a step of purifying and/or enriching cells, preferably in whole blood and/or non-trypsinized tissue.
1. A method for producing and detecting an amplicon from a bisulfite treated human leukemia inhibitory factor (LIF) gene region from human follicular helper T cells or activated helper T cells, the method comprising:
a) bisulfite treating isolated DNA from a human T cell sample comprising the human follicular helper T cells or activated helper T cells to thereby generate bisulfite treated DNA where unmethylated cytosines are converted to uracils,
b) producing the amplicon from the bisulfite treated DNA by amplifying with quantitative polymerase chain reaction (qPCR) a region of the bisulfite treated DNA comprising SEQ ID NO: 1 and having bisulfite convertible cytosine at one or more cytosine-phosphate-guanine (CpG) positions in SEQ ID NO: 1 prior to bisulfite treatment, and
c) detecting the amplicon with a probe comprising the nucleic acid sequence of SEQ ID NO: 4.
2. The method according to claim 1 , wherein said at least one CpG position is present in the 5′ region upstream from a transcription start, promoter region, the 5′ or 3′ untranslated regions, exon, intron, exon/intron border and/or in the 3′ region downstream of a transcriptional stop of said gene.
3. The method according to claim 1 , wherein the CpG position corresponds to any one of CpG positions in SEQ ID NO: 1.
4. The method according to claim 1 , further comprising detecting the bisulfite convertible cytosine using a method selected from a methylation specific enzymatic digest; bisulfite sequencing; an analysis selected from promoter methylation, CpG island methylation, MSP, HeavyMethyl, MethyLight, and Ms-SNuPE; and other methods relying on a detection of amplified DNA.
5. The method according to claim 1 , wherein said sample is a body fluid, tissue, organ, or cell.
6. The method according to claim 1 , wherein said method is performed without a step of purifying and/or enriching said cells.
7. The method according to claim 1 , wherein said sample is from a subject suffering from or is likely to suffer from an autoimmune disease, transplant rejection, infection, cancer, and/or an allergy.
8. The method of claim 1 performed using a kit, comprising a) a bisulfite reagent, and b) materials for detecting the bisulfite convertible cytosine at the at least one CpG position.
9. The method according to claim 1 , wherein the amplifying comprises using an oligomer comprising a nucleic acid sequence of any of SEQ ID NOs: 2 to 4.
10. The method according to claim 3 , wherein the CpG position is selected from CpG positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, and 23 of SEQ ID NO: 1.
11. The method, according to claim 5 , wherein the sample is a blood sample.
12. The method, according to claim 5 , wherein the sample is whole blood and/or non-trypsonized tissue.
13. The method according to claim 8 , wherein the materials for detecting the bisulfite convertible cytosine comprise an oligo selected from the sequences according to SEQ ID NOs: 2 to 4.
14. The method according to claim 1 , wherein the human T cell sample is isolated from a human blood sample.
15. The method of claim 1 , comprising
d) detecting methylation status of the one or more cytosine-phosphate-guanine (CpG) positions from the amplicon.
16. The method of claim 1 , wherein the qPCR is performed with methylation specific primers.
17. The method of claim 1 , wherein the one or more CpG positions comprising the bisulfite convertible cytosine is selected from CpG positions 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21 in SEQ ID NO: 1.
18. The method of claim 1 , wherein SEQ ID NO: 1 comprises a bisulfite convertible cytosine at CpG positions 11, 12, 13, 14, 15, 16, 17, 18, and 19 relative to SEQ ID NO: 1.