Rapid and efficient generation of antigen-specific isogenic T cells from cryopreserved blood samples
| Autor: | Floris Foijer, N. van Rooij, AL Eerkens, M. de Bruyn, Annege Vledder, Hans W. Nijman |
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| Přispěvatelé: | Stem Cell Aging Leukemia and Lymphoma (SALL), Restoring Organ Function by Means of Regenerative Medicine (REGENERATE), Translational Immunology Groningen (TRIGR), Targeted Gynaecologic Oncology (TARGON) |
| Rok vydání: | 2022 |
| Předmět: |
immune monitoring
proliferation medicine.medical_treatment T cell Immunology T cells knockout Biology CULTURE Mice Genome editing Antigen medicine Immunology and Allergy CRISPR Animals Cas9 Gene Editing clinical trials viability T-cell receptor ELISPOT Cell Biology Cell biology Cytokine medicine.anatomical_structure Leukocytes Mononuclear CRISPR-Cas Systems Antigen-specific immune responses Ex vivo |
| Zdroj: | Immunology and Cell Biology. Wiley |
| ISSN: | 1440-1711 0818-9641 |
| Popis: | ObjectivesCRISPR/Cas9-mediated gene editing has been leveraged for the modification of human and mouse T cells. However, limited experience is available on the application of CRISPR/Cas9 electroporation in cryopreserved T cells collected during e.g. clinical trials.MethodsPBMCs from healthy donors were used to generate knockout T cell models for interferon-γ (IFNγ), Cbl Proto-Oncogene B (CBLB), Fas cell surface death receptor (Fas) and T cell receptor (TCRαβb) genes. The effect of CRISPR-cas9-mediated gene editing on T cells was evaluated using apoptosis assays, cytokine bead arrays and ex vivo and in vitro stimulation assays.ResultsOur results demonstrate that CRISPR/Cas9-mediated gene editing of ex vivo T cells is efficient and does not overtly affect T cell viability. Cytokine release and T cell proliferation were not affected in gene edited T cells. Interestingly, memory T cells were more susceptible to CRISPR/Cas9 gene editing than naïve T cells. Ex vivo and in vitro stimulation with antigens resulted in equivalent antigen-specific T cell responses in gene-edited and untouched control cells; making CRISPR/Cas9-mediated gene editing compatible with clinical antigen-specific T cell activation and expansion assays.ConclusionHere, we report an optimized protocol for rapid, viable and highly efficient genetic modification in ex vivo human antigen specific T cells, for subsequent functional evaluation and/or expansion. Our platform extends CRISPR/Cas9-mediated gene editing for use in gold-standard clinically-used immune-monitoring pipelines and serves as a starting point for development of analogous approaches such as those including transcriptional activators and or epigenetic modifiers. |
| Databáze: | OpenAIRE |
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