Abstract 2187: Epigenetic monitoring of gamma delta T-cells
Authors: Olek Sven , Baron Udo , Janack Isabell , Bourquain Kati , Kärst Stefan , Phippard Deborah , Lozza Laura , Rosa Ciro Novaes Lino 
Published: April 2023

Abstract
Human gamma delta T cells (γδ-T cells) are non-conventional T lymphocytes expressing a TCR heterodimer comprised of gamma (γ) and delta (δ) chains. They constitute an essential part of the innate immune system playing a pivotal role at the front line of the host's defense against pathogens. γδ-T cells have received particular attention as they exert multiple functions under non-infectious settings, most notably in carcinogenesis, where they can initiate a potent HLA-independent anti-tumor activity. Owing to these attributes, γδ-T cells appear to be a promising target for designing cell-based anti-cancer immunotherapies. For such strategies - which involve manipulation of γδ-T cells, e.g. by activation, tumor-targeting, or genetic modification - accurate and quantitative cell-counting tools are indispensable. The gold standard for immune cell quantification to date is still flow cytometry (FCM), although this technology is associated with demanding sample logistics as it requires suspensions of viable cells and presents challenges to standardization amongst different sites, cytometers and operators. In recent years, a huge body of evidence has been accumulated demonstrating epigenetic qPCR (Epiontis ID®) as a powerful and FCM-equivalent methodology for cell quantification and immune monitoring, which is not affected with such limitations. Here we present a DNA methylation-specific, real-time PCR-based assay for the precise quantification of γδ-T cells in blood, tissues and formalin fixed and paraffin embedded (FFPE) samples. The qPCR assay targets a differentially methylated region in the gene encoding the constant region of the T cell receptor gamma chain (TRGC2). This marker region was shown being entirely unmethylated in γδ-T cells, but appearing fully methylated in conventional CD4+ and CD8+ T cells, in CD56+ NK and CD19+ B cells, in CD14+ monocytes and CD15+ granulocytes. Validation of the assay according to ICH guidelines and ISO17025 standards demonstrated a robust performance and stability, linear measurements in the operational range (between 0% and 100% methylation), a definite target cell type specificity and a good correlation to paralleled flow cytometric analyses. The intra- and inter-assay precision is below 15% and 20%, respectively and the lower limit of quantification, above where standard precision of measurements can be achieved, lies at 74 unmethylated target molecules. Cell type-specificity was shown using blood from heathy donors, the applicability in cancerous samples still has to be confirmed.In summary the epigenetic, γδ-T cell-specific assay posted here may represent a useful tool for immune monitoring in clinical settings assisting the development of innovative γδ-T cell-based immunotherapies.
Citation Format: Sven Olek, Udo Baron, Isabell Janack, Kati Bourquain, Stefan Kärst, Deborah Phippard, Laura Lozza, Ciro Novaes Lino Rosa. Epigenetic monitoring of gamma delta T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2187.