ERGIC1 as epigenetic marker for the identification of immune cells, in particular monocytic myeloid-derived suppressor cells (mMDSCs)
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Publication Number
US-11976327-B2
Publication Date
2024-05-07
Expiration Date
Abstract
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.
Core Innovation
The disclosure describes using ERGIC1 (SEQ ID NO: 1) epigenetic methylation/demethylation patterns as a marker to identify and quantify monocytic myeloid-derived suppressor cells (mMDSCs) in human cell samples. The monocytic mMDSC phenotype is CD15−CD14+CD33+CD11b+HLADR+, and the marker is based on the presence of bisulfite-convertible cytosines versus non-convertible cytosines associated with ERGIC1 sequence features.
A method is provided in which isolated genomic DNA from the CD15−CD14+CD33+HLADR+ monocytic mMDSC-containing sample is bisulfite treated and an ERGIC1 gene region is amplified to generate an amplicon. The amplicon is characterized by cytosine-adenine (CA) at defined CpG positions relative to SEQ ID NO: 1, including nucleotide positions 56, 62, and 71, and additional ERGIC1 amplicon variants are described using SEQ ID NO: 2 and SEQ ID NO: 3 with specific CA patterns at defined nucleotide positions.
The disclosure further supports quantification based on ratios of bisulfite-convertible versus non-convertible cytosines, and it distinguishes target monocytic mMDSCs from non-MDSC and other immune cell types in complex samples without purification/enrichment. Primer/probe sets associated with specific ERGIC1 amplicons (SEQ ID NOs: 4–11) are included, with example results reporting high detection specificity and linearity for target mMDSCs versus other immune cell types in the presence of complex sample backgrounds.
In addition to amplicon production and measurement, kit and monitoring/use claims are described for immune status assessment in clinical contexts, based on the ERGIC1 methylation-status marker approach and the quantified identification of monocytic mMDSCs.
Claims Coverage
The document provides three independent claims focused on producing an ERGIC1 amplicon from bisulfite treated isolated genomic DNA of CD15−CD14+CD33+HLADR+ monocytic mMDSCs, with CA/CpG position constraints on specific ERGIC1 sequence definitions (SEQ ID NO: 1 or SEQ ID NO: 3). Across the independent claims, the inventive features are defined by a combination of sample specification, bisulfite treatment, ERGIC1 region selection, PCR amplification, and specific CA at defined nucleotide positions.
Bisulfite treating CD15−CD14+CD33+HLADR+ monocytic mMDSC genomic DNA and amplifying ERGIC1 region containing CA at defined CpG positions
Bisulfite treating isolated genomic DNA from a human cell sample comprising cluster of differentiation (CD) 15−CD14+CD33+CD11b+ HLADR+ monocytic myeloid-derived suppressor cells (mMDSCs) to generate bisulfite treated DNA, and amplifying from the bisulfite treated DNA a region of ERGIC1 gene comprising SEQ ID NO: 1 to thereby generate the amplicon, wherein the amplicon comprises a cytosine-adenine (CA) at at least CpG positions at nucleotides 56, 62, and 71 relative to SEQ ID NO: 1.
Producing ERGIC1 amplicon with nucleotides 46-148 of SEQ ID NO: 3 and CA at defined nucleotide positions
Bisulfite treating isolated genomic DNA from a human cell sample comprising CD15−CD14+CD33+CD11b+ HLADR+ monocytic mMDSCs to generate bisulfite treated DNA where unmethylated cytosines are converted to uracils, and amplifying with polymerase chain reaction (PCR) a region of the bisulfite treated DNA to produce an amplicon comprising nucleotides 46-148 of SEQ ID NO: 3 and having cytosine-adenine (CA) at nucleotide positions 56, 62, and 71 relative to SEQ ID NO: 3.
Producing ERGIC1 amplicon with nucleotides 46-148 of SEQ ID NO: 3 and CA at a reduced set of nucleotide positions
Bisulfite treating isolated genomic DNA from a human cell sample comprising CD15−CD14+CD33+CD11b+ HLADR+ monocytic mMDSCs to generate bisulfite treated DNA where unmethylated cytosines are converted to uracils, and amplifying with polymerase chain reaction (PCR) a region of the bisulfite treated DNA to produce an amplicon comprising nucleotides 46-148 of SEQ ID NO: 3 and having cytosine-adenine (CA) at nucleotide positions 62 and 71 relative to SEQ ID NO: 3.
Across the independent claims, the core claim coverage centers on generating ERGIC1 amplicons from bisulfite treated isolated genomic DNA obtained from CD15−CD14+CD33+CD11b+HLADR+ monocytic mMDSCs, with CA/CpG presence constrained to specified CpG/nucleotide positions relative to defined ERGIC1 sequence references (SEQ ID NO: 1 and SEQ ID NO: 3) after PCR amplification.
Stated Advantages
Distinguishes target monocytic mMDSCs from non-MDSC and other immune cell types in complex samples without purification/enrichment.
Quantification is supported by ratios of bisulfite-convertible versus non-convertible cytosines using ERGIC1 amplicon variants and defined primer/probe sets.
Example results report substantially higher detection for target mMDSCs versus other immune cells.
Documented Applications
Immune status assessment and monitoring in clinical contexts based on ERGIC1 methylation-status marker measurement for identification and quantification of monocytic mMDSCs.
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