Precision for Medicine Gmbh

Epiontis Gmbh

Leland Stanford Junior University

Charité – Universitätsmedizin Berlin

Sven Olek

Udo Baron

Ulrich Hoffmüller

Ivana Türbachova

Rosa Bacchetta

Jochen Huehn

Stefan Floess

Alf Hamann

Ulrich Hoffmueller

Paul Gardina

Janika Josephin Schulze

Josephin Held

Tim Schwachula

Ivana Tuerbachova

Jochen Hühn

Stefan Flöss

The present invention relates to a method, in particular an in vitro method, for specifically identifying the C-X-C motif chemokine receptor 3 (CXCR3) subpopulation of immune cells in a sample from a mammal comprising immune cells, in particular at least one of CD8+ effector and memory T cells, T helper (Th)1 cells and natural killer T (NKT) cells, comprising analyzing epigenetic modifications/properties of (including the methylation status) of at least one CpG position in the mammalian gene region for CXCR3 according to SEQ ID No. 1, wherein a demethylation or lack of methylation of said of at least one CpG position in said gene region is indicative for said CXCR3 specific subpopulation of immune cells in said sample, when compared to other immune cells, in particular for at least one of CD8+ effector and memory T cells, T helper (Th)1 cells and natural killer T (NKT) cells. The analyses according to the invention can identify the above 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 the above cells, in particular 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.

CXCR3 as epigenetic marker for the identification of inflammatory immune cells, in particular CD8+ memory t cells

The present invention relates to a method, in particular an in vitro method, for identifying CD3CD4 positive T lymphocytes of a mammal, wherein said method comprises analysing the methylation status of at least one CpG position in the CD3a/b/c/d/g genes, in particular their 'upstream' regulatory regions, and in particular the promoter and other conserved regions of the gene cd3, wherein a demethylation of at least one CpG in the analyzed sample to at least 90% is indicative for memory and naive CD4 or/and memory and/or naive T lymphocytes. Furthermore, the present invention is directed at the use of DNA-methylation analysis of the genes CD3a/b/c/d for the detection and quality assurance and control of T lymphocytes. Furthermore, the present invention relates to a kit for performing the above methods as well as respective uses thereof. In a preferred embodiment, the present invention furthermore provides an improved method for analysing the methylation status of at least one CpG position in the gene CD3, allowing for a precise analysis even from sub-optimal quality samples, such as non-freshly obtained blood or serum samples.

Epigenetic markers for the identification of blood sub-cells of type 1

The present invention relates to an epigenetic method for identifying IPEX (immunodysregulation polyendocrinopathy enteropathy X-linked) syndrome and/or IPEX-like syndrome in a human subject using the methylation status of F0XP3 and a control gene specific for a CD4 and/or CD3 T cell. The method is used to distinguish IPEX from IPEX-like and from IPEX-unrelated disorders.

Epigenetic method to detect and distinguish IPEX and IPEX-like syndromes, in particular in newborns

The present invention relates to improved methods for epigenetic blood and immune cell detection and counting, and respective uses and kits.

Method for epigenetic immune cell detection and counting in human blood samples

The present invention relates to a method, in particular an in vitro method, for identifying specific immune cells, in particular MDSCs, comprising analyzing a modification, preferably the methylation status, of at least one CpG position in the mammalian gene region for endoplasmatic reticulum-golgi intermediate compartment 1 (ERGIC1), wherein a demethylation or lack of methylation or modification of said gene region is indicative for an MDSC, when compared to a non-MDSC. The analyses according to the invention can identify specific sub-populations of MDSCs, namely monocytic MDSCs (m MDSCs) 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 m MDSCs, in particular 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.

ERGIC1 as epigenetic marker for the identification of immune cells, in particular monocytic myeloid-derived suppressor cells (mMDSCs)

The present invention relates to a method, in particular an in vitro method, for identifying CD56+ NK cells, comprising analyzing the methylation status of at least one CpG position in the mammalian gene region for mevalonate (diphospho) decarboxylase (MVD), wherein a demethylation or lack of methylation of said gene region is indicative for a CD56+ NK cell, when compared to a non-CD56+ NK cell. The analyses according to the invention can identify CD56+ NK 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 CD56+ NK cells, in particular 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.

MVD as epigenetic marker for the identification of immune cells, in particular CD56+ NK cells

The present invention relates to a method, in particular an in vitro method, for identifying monocytes, comprising analyzing the methylation status of at least one CpG position in the mammalian gene region for parkin RBR E3 ubiquitin protein ligase (PARK2), wherein a de-methylation or lack of methylation of said gene region is indicative for a monocyte, when compared to a non-monocyte cell. The analyses according to the invention can identify monocytes 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 monocytes, in particular 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. Also claimed are kits and specific oligonucleotides for use as primers or probes.

PARK2 as epigenetic marker for the identification of immune cells, in particular monocytes

The present invention relates to a method, in particular an in vitro method, for identifying basophil granulocytes, comprising analyzing a modification such as for example the methylation status of at least one CpG position in the mammalian gene region for the gene 'mutated in colorectal cancer' (MCC), wherein a demethylation or lack of modification or methylation of said gene region is indicative for a basophil granulocyte, when compared to a non-basophil granulocyte, or any other cell type in the peripheral blood or in other tissues. The analyses according to the invention can identify basophil granulocytes 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 basophil granulocytes, in particular in 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. Finally, the invention provides primers, probes and amplicons based on bisulfite treated nucleic acids.

MCC as epigenetic marker for the identification of immune cells, in particular basophil granulocytes

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.

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 non-classical monocytes, comprising analyzing the methylation status of at least one CpG position in the mammalian genomic region comprising an amplicon, wherein a demethylation or lack of methylation of said region is indicative for a non-classical monocyte, when compared to a classical monocyte or a non-monocyte cell. The analyses according to the invention can identify non-classical monocytes 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 non-classical monocytes, in particular 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.

Amplicon region as epigenetic marker for the identification of non-classical monocytes

The present invention relates to a method, in particular an in vitro method, for identifying specific immune cells, in particular na&#xef;ve 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 na&#xef;ve CD8+ T-cell, when compared to a non-na&#xef;ve CD8+ T-cell or any other (blood) cell type. The analyses according to the invention can identify na&#xef;ve 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 na&#xef;ve CD8+ T-cells, in particular in com na&#xef;ve 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.

Endosialin (CD248) as epigenetic marker for the identification of immune cells, in particular na&#xef;ve CD8+ t-cells

The present invention relates to a method, in particular an in vitro method, for identifying B cells, comprising analyzing the methylation status of at least one CpG position in the mammalian gene region for Low density lipoprotein receptor-related protein 5 (LRP5), wherein a demethylation or lack of methylation of said gene region is indicative for a B cell, when compared to a non-B cell. The analyses according to the invention can identify B cells on an epi-genetic 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 B cells, in particular 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. Further claimed are kits and specific primers and probes for identifying methylation.

LRP5 as epigenetic marker for the identification of immune cells, in particular B-cells

The present invention relates to a method, in particular an in vitro method, for identifying PD1+ cells, comprising analyzing the methylation status of at least one CpG position in the mammalian gene region for Programmed cell death 1 (PDCD1), wherein a demethylation or lack of methylation of said gene region is indicative for a PD1+ cell, when compared to a non-PD1+ cell. The analyses according to the invention can identify PD1+ 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 PD1+ cells, in particular 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.

PDCD1 as epigenetic marker for the identification of immune cells, in particular PD1+ cells

The present invention relates to improved methods for epigenetic blood and immune cell counting, and respective uses and kits.

Method for epigenetic immune cell counting

The present invention provides an epigenetic haemogram, also referred to as an epigenetic blood cell count that identifies the quantitative, comprehensive picture of cellular composition in a biological sample, wherein advantageously a normalization standard is used. The normalization standard is a nucleic acid molecule comprising at least one marker-region being specific for each of the blood cells to be detected, and at least one control-region being cell-unspecific, wherein said regions are present in the same number of copies on said molecule and/or a natural blood cell sample of known composition. Furthermore, the present invention relates to a kit and the use of a kit for performing the epigenetic assessment of comprehensive, quantitative cellular composition of a biological sample. The biological sample is derived from e.g. a mammalian body fluid, including peripheral, capillary or venous blood samples or subfractions thereof, such as peripheral blood mononuclear cells or peripheral blood monocytes, or a tissue sample, organ sample, or from frozen, dried, embedded, stored or fresh body fluids or tissue samples.

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Berlin, Germany

Sven Olek