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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.

Tumor-Infiltrating (TI) T cells play an important role in anti-tumor immunity, highlighted by multiple immunotherapy strategies approved by the FDA. However, the benefit of current immunotherapies is still limited, such that additional research is needed to improve efficacy. Preclinical tumor models are essential to advance immunotherapy research. However, tumor composition is highly variable and T cell numbers can be very limited. Even flow cytometry can be very time consuming. Importantly, TI T cells are embedded in a cellular environment where antigen is abundant and surrounding cells express immunomodulatory molecules, such that unbiased cell-intrinsic functional characterization is impossible. Pre-enrichment of TI T cells is highly desirable to reduce hands-on time, and generate high quality and reliable data. We developed new CD4, CD8 and Pan T cell specific enrichment reagents for magnetic cell sorting from mouse tumors. Single cell suspension was obtained by automated dissociation. T cell isolation was done either manually, or using semi or full automation. TI T cell frequencies ranged from 0.2–9%, were isolated to purities above 80% with yields ranging from 60–95%. Pan T cell isolation maintained the original CD4:CD8 ratio. CD69 was not upregulated after 18h culture, demonstrating that isolation did not result in T cell activation. Importantly, functional analysis of bulk vs. purified cells revealed significant influence of tumor and stromal environment in cytokine expression by TI T cells, which was not due to the isolation protocol. Our data, demonstrates that magnetic enrichment reduces time of analysis, increases quality of analysis and is essential for the functional characterization of TI T cells.

T cells can eliminate tumors, but tumor infiltrating (TI) T cells can become intrinsically anergic or extrinsically suppressed. Identifying the mechanisms of T cell dysfunction is necessary to improve immunotherapy efficacy. Mouse tumor models are useful tools in tumor T cell research, but analysis of TI T cells is technically challenging, time consuming and labor intensive. Their frequency can be very low and small populations may escape analysis as they get lost in the background. Importantly, TI T cells are in an antigen-rich and highly immunomodulatory cell environment, such that cell intrinsic functional analysis is hindered. It is fundamental to use new tools to streamline the workflow and generate reliable data. We established workflows combining tissue dissociation, T cell isolation and phenotyping. Tumor dissociation was automated using the gentle MACS™ and optimized for epitope preservation. Enzymatic dissociation was essential for analysis of PD1hiTim3+Lag3+CD39+ CD8 T cells in tumors. Using new T cell-specific microbeads for magnetic cell sorting we increased TI T cell purity by up to 500-fold, while maintaining activation status and phenotype. Importantly T cell isolation from the tumor’s highly immunomodulatory cellular environment was essential to assess cell-intrinsic functional properties. Finally, our data show the use of recombinant RE Afinity antibodies eliminated non-specific labeling of cells in the tumor microenvironment. We conclude that standardized processing of tumor samples and magnetic isolation of tumor infiltrating T cells greatly reduces time and cost of total workflow and downstream analysis while significantly increasing reproducibility and the quality of data obtained from TIL analysis.