Naïve CD8+ T-cell Identification via CD248 Methylation Analysis
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Solution Overview
Problem
Current methods for identifying and quantifying naïve CD8+ T-cells are not robust enough, particularly in complex samples, and often require purification and staining procedures, which can be cumbersome and inefficient.
Innovation Solution
Analyzing the methylation status of specific CpG positions in the CD248 gene region, specifically using bisulfite conversion and PCR techniques, allows for the identification and quantification of naïve CD8+ T-cells without the need for purification or staining, utilizing specific primer pairs and amplicons to differentiate them from other immune cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current methods for identifying naïve CD8+ T-cells are used, then identification can be performed, but the methods are not robust enough in complex samples and require purification and staining procedures
Solution Approach 1:
The invention extracts and utilizes the specific epigenetic signature (methylation pattern) of naïve CD8+ T-cells from complex samples. By focusing on the unique demethylated state of the CD248 gene in this cell population, the method extracts the essential identifying feature without requiring physical separation or staining of cells, thereby simplifying the procedure while maintaining high identification reliability
Solution Approach 2:
The invention replaces mechanical/physical separation methods (purification and staining procedures) with a molecular biology-based detection method. Instead of physically isolating and visually identifying cells through staining, the method uses PCR-based detection of methylation patterns to identify naïve CD8+ T-cells directly in complex samples, eliminating the need for cumbersome purification and staining steps
2Ease of operation
If purification and staining procedures are used to identify naïve CD8+ T-cells, then cell identification is possible, but the procedures are cumbersome and inefficient
Solution Approach 1:
The invention extracts the essential identifying feature (epigenetic signature) from the complex cellular system, allowing direct detection of naïve CD8+ T-cells through their unique methylation pattern. This extraction of the key identifying characteristic eliminates the need for multiple procedural steps including purification and staining, making the method both simpler and more efficient
Solution Approach 2:
The invention changes the detection parameter from surface protein expression (detected by staining) to epigenetic modification state (detected by methylation analysis). This parameter change allows identification of naïve CD8+ T-cells through their demethylated CD248 gene, which can be detected directly in complex samples without purification or staining, thereby simplifying the procedure and improving efficiency
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method provides a robust and reliable means to detect and quantify naïve CD8+ T-cells in various samples, including whole blood and tissues, with high specificity and sensitivity, enabling their identification in autoimmune diseases, cancer, and other immune-related conditions.
Implementation Method 1
analyzing the modification, such as the methylation status, of at least one CpG position in the mammalian gene region for endosialin (CD248)
Implementation Method 2
specifically using bisulfite conversion and PCR techniques
Data Source
AI summary
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.