Identification and Quantification of Immune Cell types using PCR-based PureQuant Methylation Assay

Background & Aim Development and manufacturing of cell based immunotherapies rely on methods that can accurately identify and quantify cell purity. Current methods predominantly rely on the detection of biomarkers using flow cytometry. These methods, while effective for the detection of surface antigens such as CD8+ that positively identifies Cytotoxic T lymphocytes, are more challenging to implement for intracellular markers; for example those that are used to positive identify Regulatory T (Treg) cells and T Helper 17 (Th17) cells. Additionally, characterization methods that utilize live cells pose a challenge in large scale GMP manufacturing environment due to complicated logistics, limited throughput and can be difficult to standardize. Therefore, there is an emerging need for alternative assay methods that address some of these challenges. Methods, Results & Conclusion Here, we report the development of PureQuant Methylation Assays that specifically identifies and quantifies immune cell types based on their unique DNA methylation status. Percentage of CD8, Treg and Th17 was determined by detecting methylation status specific for cytotoxic T cells, FoxP3 and IL17A respectively, via qPCR of bisulfite converted genomic DNA. Unlike flow cytometry, methylation workflow for surface and intracellular markers are similar and avoid the need for stimulation for the detection of low marker levels such as Th17. These assays require minimal sample, can use fresh/frozen cells or isolated gDNA and can be carried out on any standard qPCR instrument. The combination of accuracy, low sample requirement and flexibility provides an ideal measurement system for confirmation of identify and purity of T cell types critical for therapeutic applications. Development and manufacturing of cell based immunotherapies rely on methods that can accurately identify and quantify cell purity. Current methods predominantly rely on the detection of biomarkers using flow cytometry. These methods, while effective for the detection of surface antigens such as CD8+ that positively identifies Cytotoxic T lymphocytes, are more challenging to implement for intracellular markers; for example those that are used to positive identify Regulatory T (Treg) cells and T Helper 17 (Th17) cells. Additionally, characterization methods that utilize live cells pose a challenge in large scale GMP manufacturing environment due to complicated logistics, limited throughput and can be difficult to standardize. Therefore, there is an emerging need for alternative assay methods that address some of these challenges. Here, we report the development of PureQuant Methylation Assays that specifically identifies and quantifies immune cell types based on their unique DNA methylation status. Percentage of CD8, Treg and Th17 was determined by detecting methylation status specific for cytotoxic T cells, FoxP3 and IL17A respectively, via qPCR of bisulfite converted genomic DNA. Unlike flow cytometry, methylation workflow for surface and intracellular markers are similar and avoid the need for stimulation for the detection of low marker levels such as Th17. These assays require minimal sample, can use fresh/frozen cells or isolated gDNA and can be carried out on any standard qPCR instrument. The combination of accuracy, low sample requirement and flexibility provides an ideal measurement system for confirmation of identify and purity of T cell types critical for therapeutic applications.