T cells play a central role in immunity to pathogens and tumors, but also in autoimmunity. T cell research has generated knowledge that led to multiple clinical breakthroughs, such as the generation of T cell vaccines and tumor immunotherapy approaches. However, additional work is required to fully understand T cell biology, harness their therapeutic potential and control immunopathologies. Cutting edge experimental protocols that interrogate and manipulate T cell biology often require prior purification of T cell populations. Strategies to simplify and accelerate T cell purification are highly desirable to save time, reduce bias and allow complex experiments to be performed. We established workflows combining automated tissue dissociation with T cell isolation. We developed new CD4, and CD8 T cell specific enrichment reagents that significantly accelerated the isolation protocol. T cells were isolated to purities above 90%, reducing time of downstream analysis. Importantly, culture of isolated T cells did not induce activation as measured by CD69 or CD25 upregulation, as compared to controls. Furthermore, magnetic isolation did not affect proliferation or cytokine expression following in vitro stimulation. Finally, T cell isolation could be fully automated and multiple samples could be processed in parallel. Our new workflow greatly reduces time of downstream analysis while preserving cell phenotype and functional properties. We believe these innovative tools significantly shorten time-consuming experiments and can be used to increase reproducibility and the quality of data obtained in T cell research.
T cells play a central role in immunity to pathogens and tumors, but also in autoimmunity. T cell research has generated knowledge that led to multiple clinical breakthroughs, such as the generation of T cell vaccines and tumor immunotherapy approaches. However, additional work is required to fully understand T cell biology, harness their therapeutic potential and control immunopathologies. Cutting edge experimental protocols that interrogate and manipulate T cell biology often require prior purification of T cell populations. Strategies to simplify and accelerate T cell purification are highly desirable to save time, reduce bias and allow complex experiments to be performed. We established workflows combining automated tissue dissociation with T cell isolation. We developed new CD90.1, and CD90.2 T cell specific enrichment reagents that significantly accelerated the isolation protocol. These reagents could be used to isolate total T cells, or rare CD90.1+ or CD90.2+ cells from adoptive transfer models. Importantly, culture of isolated T cells did not induce activation as measured by CD69 or CD25 upregulation, as compared to controls. Furthermore, magnetic isolation did not affect proliferation or cytokine expression following in vitro stimulation. Finally, T cell isolation could be fully automated and multiple samples could be processed in parallel. Our new workflow greatly reduces time of downstream analysis while preserving cell phenotype and functional properties. We believe these innovative tools significantly shorten time-consuming experiments and can be used to increase reproducibility and the quality of data obtained in T cell research.
BackgroundAdoptive cell transfer of chimeric antigen receptor (CAR) modified T cells has demonstrated great therapeutic success against certain hematological malignancies. However, a substantial number of patients experienced relapse at some point after treatment with the underlying mechanisms not fully understood. Emerging data suggest that the undesired clinical outcome is related to different aspects, which include: the tumor heterogeneity, the tumor microenvironment, as well as intrinsic characteristics of the CAR T cells. In this work, we aimed to understand the diversity of CAR T cells generated from different donors, using multiparameter in vitro characterization.MethodsLeukapheresis from healthy donors were collected to generate CAR T cells using the GMP-compliant CliniMACS Prodigy® platform, enabling an automated and closed engineering of CAR T cells in a highly reproducible manner. We performed an in-depth characterization of the resulting CAR T cells by exploring differences in the immunophenotype, cell fitness and effector function of the freshly prepared as compared to frozen CAR T cell samples. Specifically, we designed several flow cytometry panels for the extensive characterization of immunophenotypes of interest such as: proliferative capacity, differentiation, activation and exhaustion. Cell fitness status was determined by the rate at which cells undergo apoptosis following stress. Finally, effector function was determined by the ability of the activated CAR T cells to secrete proinflammatory cytokines including IFN-g, TNF-a and IL-2. The associations between these different parameters were analyzed using comprehensive statistical approaches.ResultsWith our established workflow, over 20 healthy-donor derived CAR T cells were generated and characterized. We have observed donor-dependent variations and responses for most of the explored parameters. In general, the freezing and thawing process negatively affected cell fitness and effector function of the CAR T cells and resulted in altered immunophenotypes. Additionally, correlations between certain immunophenotypes and cell fitness/effector function were identified.ConclusionsCollectively, we established a workflow for multiparameter characterization of CAR T cells and assessed the intrinsic variability of CAR T cells for both research and clinical application.
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