Activation-neutral gene editing of tonsillar CD4 T cells for functional studies in human ex vivo tonsil cultures

Abstract

The molecular and immunological properties of tissue-resident resting CD4 Tcells are understudied due to the lack of suitable gene editing methods. Here, we describe the exvivo culture and gene editing methodology ediTONSIL for CD4 Tcells from human tonsils. Optimized CRISPR-Cas9 RNP nucleofection results in knockout efficacies of over 90% without requiring exogenous activation. Editing can be performed on multiple cell types in bulk cultures or on isolated CD4 Tcells that can be labeled and reintroduced into their tissue environment. Importantly, CD4 Tcells maintain their tissue-specific properties such as viability, activation state, or immunocompetence following reassembly into lymphoid aggregates. This highly efficient and versatile gene editing workflow for tonsillar CD4 Tcells enables the dissection of molecular mechanisms in exvivo cultures of human lymphoid tissue and can be adapted to other tonsil-resident cell types.