REAlease® Immunomagnetic Separation Technology with Reversible Labeling for Tumor Infiltrating T Cells and Sub-Populations

Abstract Immunotherapies engaging T cells have proven clinical efficacy and tremendous potential. However, responses are often suboptimal. Further research is required to understand tumor-infiltrating leukocytes (TILs) biology and enhance outcomes. TIL analysis is technically challenging and labor intensive. Their number can be very low and small subpopulations may escape analysis as they get lost in the background noise. Importantly, tumor-infiltrating (TI) T cells are embedded in a highly immunomodulatory environment such that unbiased cell-intrinsic functional characterization is hindered. When working with large cohort sizes, even immunophenotyping TILs by flow cytometry is time consuming and data processing is laborious. Pre-enrichment of TI T cells is highly desirable to reduce hands-on time and generate high quality and reliable data. We developed state-of-the-art CD4, CD8, CD3 and CD4/CD8 T cell specific enrichment reagents for magnetic cell sorting incorporating novel technology enabling the removal of both superparamagnetic beads and antibody fragments (REAlease®). Single cell suspension of human ovarian, pancreas or colon tumors was obtained by automated dissociation. TI T cell frequencies ranged from 0,5–14%, were isolated to high purities and with high yield. CD69 was not upregulated after 18h culture, and cells were functional for downstream applications. Importantly, REAlease technology allowed secondary magnetic isolation of sub-populations of T cells, such as gamma-delta T cells. Our data demonstrates that magnetic enrichment using REAlease technology reduces time and increases quality of analysis, and is essential for the isolation of important T cell sub-populations within the tumor microenvironment.