Epigenetic method for the identification of follicular T-helper-(TFH-) cells

Inventors

Olek, Sven • HOFFMÜLLER, Ulrich

Assignees

Precision for Medicine GmbH

Abstract

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.

Core Innovation

The invention relates to an in vitro method for producing and detecting an amplicon from a bisulfite treated human leukemia inhibitory factor (LIF) gene region. The method uses human follicular helper T cells or activated helper T cells as the source of DNA and relies on bisulfite converting unmethylated cytosines into uracils, thereby generating bisulfite treated DNA with a methylation-dependent change. The approach focuses on a region that comprises SEQ ID NO: 1 and includes bisulfite convertible cytosine at one or more cytosine-phosphate-guanine (CpG) positions in SEQ ID NO: 1 prior to bisulfite treatment.

The produced amplicon is generated by quantitative polymerase chain reaction (qPCR) and detected using a defined probe. Detection is based on amplifying a CpG-containing portion of the bisulfite treated LIF gene region and measuring the resulting amplicon signal using a probe comprising the nucleic acid sequence of SEQ ID NO: 4. The method is described as enabling analysis of methylation/bisulfite convertibility at CpG positions in the LIF region, so that demethylation at the selected CpG positions corresponds to bisulfite convertibility and methylation corresponds to non-convertibility.

The document further describes the method as usable without cell purification/enrichment steps in complex samples such as whole blood. It reports an example qPCR assay with primer/probe sequences and measured TFH copy-number/percentage across donors, along with a proposed quantification based on the ratio of bisulfite-convertible (demethylated) LIF to methylated (non-convertible) LIF. Optional normalization to control genes such as GAPDH is described to support quantification.

Claims Coverage

The patent includes one independent method claim and its dependents. The independent claim covers producing and detecting a qPCR amplicon from a bisulfite treated human LIF gene region from human follicular helper T cells or activated helper T cells, using defined SEQ ID NO: 1 and a probe defined by SEQ ID NO: 4, with bisulfite conversion of unmethylated cytosines as the methylation-dependent basis for detection.

Across the independent claim, the inventive concept is the combination of bisulfite conversion on DNA from human follicular helper T cells or activated helper T cells, qPCR amplification of a defined SEQ ID NO: 1 LIF gene region containing bisulfite convertible CpG positions, and detection of the resulting amplicon using a probe comprising SEQ ID NO: 4.

Stated Advantages

Documented Applications