Endosialin (CD248) as epigenetic marker for the identification of immune cells, in particular naïve CD8+ t-cells
The present invention relates to a method, in particular an in vitro method, for identifying specific immune cells, in particular naïve CD8+ T-cells, comprising analyzing the methylation status of at least one CpG position in the mammalian gene region for endosialin (CD248), wherein a demethylation or lack of methylation of said gene region is indicative for a naïve CD8+ T-cell, when compared to a non-naïve CD8+ T-cell or any other (blood) cell type. The analyses according to the invention can identify naïve CD8+ T-cells on an epigenetic level and distinguish them from all other cells in complex samples, such as, for example, other blood or immune cells. The present invention furthermore provides an improved method for quantifying naïve CD8+ T-cells, in particular in com naïve CD8+ T-cells complex samples. The method can be performed with or without a step of purifying and/or enriching cells, preferably in whole blood and/or non-trypsinized tissue.
1. A method for producing an amplicon from a human endosialin (CD248) gene, the method comprising:
a) bisulfite treating isolated genomic DNA from a human immune cell sample to generate bisulfite treated DNA, and
b) amplifying a region of the CD248 gene from the bisulfite treated DNA to thereby produce the amplicon, wherein the amplifying is performed with a polymerase chain reaction (PCR) using methylation-specific primers that amplify a region of SEQ ID NO: 2 and the amplicon comprises thymine-guanine (TG) at nucleotide positions 274 and 275, 292 and 293, and 296 and 297 of SEQ ID NO: 2.
2. The method according to claim 1 , further comprising detecting a methylation status of at least one cytosine-phosphate-guanine (CpG) position from the amplicon by a method selected from the group consisting of a methylation specific enzymatic digest, bisulfite sequencing, promoter methylation analysis, CpG island methylation analysis, MSP, HeavyMethyl, MethyLight, and Ms-SNuPE.
3. The method according to claim 1 , wherein said human immune cell sample is obtained from a body fluid, a tissue, or an organ.
4. The method according to claim 1 , wherein said method is performed without a step of purifying and/or enriching said sample.
5. The method according to claim 1 , wherein said sample is derived from a human who suffers from or is likely to suffer from autoimmune diseases, transplant rejections, infection diseases, cancer, and/or allergy.
6. The method of claim 1 , wherein the method is performed using a kit comprising:
a) a bisulfite reagent, and
b) materials for detecting a bisulfite convertible cytosine at CpG positions from the amplicon.
7. The method of claim 1 , wherein the PCR uses an oligomer comprising the sequence of any of SEQ ID NOs: 4 to 13.
8. The method according to claim 1 , wherein the amplicon comprises TG at nucleotide positions 274 and 275, 292 and 293, 296 and 297, and 319 and 320 of SEQ ID NO: 2.
9. The method according to claim 1 , wherein said method is performed using whole blood and/or non-trypsinized tissue.
10. The method according to claim 1 , wherein the amplicon comprises TG at nucleotide positions 274 and 275, 292 and 293, 296 and 297, 319 and 320, and 327 and 328 of SEQ ID NO: 2.
11. The method of claim 1 , wherein the amplicon comprises SEQ ID NO: 2.
12. The method according to claim 1 , wherein said human immune cell sample is obtained from a blood sample.
13. The method according to claim 1 , wherein said human immune cell sample is a blood cell type.
14. The method according to claim 1 , wherein said human immune cell sample comprises lymphocytes.
15. The method of claim 1 , wherein the amplicon comprises TG at nucleotide positions 243 and 244, 274 and 275, 292 and 293, 296 and 297, 319 and 320 of SEQ ID NO: 2.