CXCR3 as epigenetic marker for the identification of inflammatory immune cells, in particular CD8+ memory t cells
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Publication Number
US-12305237-B2
Publication Date
2025-05-20
Expiration Date
Abstract
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.
Core Innovation
The invention relates to detecting CXCR3-specific immune cell subsets by analyzing epigenetic methylation/convertibility status at CpG positions in the CXCR3 gene region defined by SEQ ID No. 1. The approach identifies and quantifies CD8+ effector/memory T cells, CD4+ Th1 cells, and NKT cells using DNA methylation status in the CXCR3 gene region.
The method produces an amplicon from the CXCR3 gene region after bisulfite treating isolated genomic DNA comprising SEQ ID No. 1. In the produced amplicon, TpG is detected at at least one CpG position, including CpG positions 150, 163, 170, and 184 relative to SEQ ID No. 1 prior to bisulfite treatment.
The document emphasizes robustness in complex samples such as whole blood and non-trypsinized tissue without purification/enrichment. It also supports restricting bisulfite-convertible patterns to the CXCR3 region defined by SEQ ID No. 1, including CpG and TpG target systems and reported demethylation levels across multiple immune cell types and conditions.
Claims Coverage
One independent claim is explicitly provided, directed to producing an amplicon from a CXCR3 gene region and detecting TpG corresponding to defined CpG positions after bisulfite treating genomic DNA from a specified human cell sample. The provided dependent claims refine the sample context, detection targets, detection modalities, and amplification materials.
Bisulfite treating and TpG detection at defined CXCR3 positions
A method for producing an amplicon from a gene region of the human C-X-C motif chemokine receptor 3 (CXCR3) gene, comprising bisulfite treating isolated genomic DNA comprising SEQ ID NO: 1 to generate bisulfite treated DNA from a human cell sample comprising CD8+ memory T cells; producing the amplicon by amplifying from a region of the bisulfite treated DNA comprising SEQ ID NO: 1 prior to bisulfite treatment; and detecting, in the amplicon, TpG at at least one of CpG positions 150, 163, 170, and 184 relative to SEQ ID NO: 1 prior to bisulfite treatment.
TpG detection across additional CpG position targets
The method further detects TpG in an amplicon at specified CpG positions relative to SEQ ID NO: 1 prior to bisulfite treatment.
Multiple alternative TpG/methylation detection approaches
TpG is detected using one of several specified methylation/DNA amplification detection techniques, including methylation specific enzymatic digest, bisulfite sequencing, promoter methylation analysis, CpG island methylation analysis, MSP, HeavyMethyl, MethyLight, Ms-SNuPE, or other amplified-DNA based detection methods.
Probe-based hybridization detection of the amplicon
Detecting an amplicon using a probe that contains a nucleic acid sequence hybridizing to a region of the amplicon.
No purification and/or enrichment of the human cell sample
The method is carried out without purifying and/or enriching the human cell sample.
Amplification using oligomers specified by SEQ ID Nos: 4 to 9
The method is performed by amplifying using an oligomer specified by SEQ ID NOs 4 through 9.
Across the independent claim and the provided refinements, the claimed core is focused on bisulfite-driven identification of CXCR3 epigenetic state by amplifying from a CXCR3 region defined by SEQ ID No. 1 and detecting TpG at defined CpG positions, including CpG positions 150, 163, 170, and 184. Dependent claims expand the set of CpG position targets, allow multiple TpG/methylation detection modalities, narrow detection to probe-based hybridization, specify constraints on sample handling without purification/enrichment, and constrain amplification materials to oligomers defined by SEQ ID Nos: 4 to 9.
Stated Advantages
Robust performance in complex samples such as whole blood and non-trypsinized tissue without purification/enrichment.
Documented Applications
Identification and quantification of CXCR3-specific immune cell subsets, including CD8+ effector/memory T cells, CD4+ Th1 cells, and NKT cells, using methylation/convertibility analysis at CpG positions in the CXCR3 gene region defined by SEQ ID No. 1.
Detection of methylation status in complex biological samples including whole blood and non-trypsinized tissue without purification/enrichment.
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