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• W4386070192 abstract "Motivation CD4 T cells are central players of adaptive immunity that rapidly change activation states in response to exogenous T cell receptor (TCR) stimulation. While molecular processes in activated human CD4 T cells from peripheral blood are well studied, resting CD4 T cells are refractory to gene-editing transduction and transfection methods without prior activation. Knowledge on the molecular biology of truly resting CD4 T cells is therefore lacking. We present here the culture and editing workflow ediTONSIL that allows for gene editing of tissue-resident CD4 T cells without compromising their activation state or immunological function. This will enable molecular mechanistic analyses of this cell type in their native/physiological tissue context. Summary The molecular and immunological properties of resting CD4 T cell biology are understudied due to the lack of suitable gene editing methods. Here we describe the ex vivo culture and gene editing methodology ediTONSIL for tissue-resident CD4 T cells from human tonsil tissue. CRISPR/Cas9 RNP nucleofection under optimized culture conditions and cytokine concentrations results in knock out efficacies of over 90%, which are e.g. sufficient to prevent HIV-1 infection when targeting the viral entry co-receptor CXCR4. EdiTONSIL does not impair tonsil CD4 T cell viability, activation state or immunocompetence and does not require exogenous activation. Editing can be performed on multiple cell types in bulk cultures or of CD4 T cells previously isolated from tonsils that can be added back to the non-CD4 T cell fraction post gene editing to reassemble into immunocompetent organotypic lymphoid aggregate structures. This highly efficient and versatile workflow for gene editing of tonsillar CD4 T cells enables the dissection of molecular mechanisms in ex vivo cultures of human lymphoid tissue and can be adapted to other tonsil-resident cell types. Graphical Abstract" @default.
• W4386070192 created "2023-08-23" @default.
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• W4386070192 date "2023-08-22" @default.
• W4386070192 modified "2023-10-10" @default.
• W4386070192 title "ediTONSIL: Activation-Neutral Gene Editing of CD4 T cells in Human ex Vivo Tonsil Cultures" @default.
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• W4386070192 doi "https://doi.org/10.1101/2023.08.21.554089" @default.
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