In vivo analysis of CRISPR-edited germinal center murine B cells

• Published in: Frontiers in immunology 2024-10, Vol.15, p.1473760
• Main Authors: Chege Kuria, Timothy, Schneider, Andrea, Baraka, Favoured, Wanzek, Jana, Vogg, Lisa, Brey, Stefanie, Habenicht, Katharina M, Winkler, Thomas H
• Format: Article
• Language: English
• Subjects: 40LB cells; Adoptive Transfer; Animals; B cells; B-Lymphocytes - immunology; CRISPR; CRISPR-Cas Systems; Fas; fas Receptor - genetics; Gene Editing - methods; germinal center; Germinal Center - immunology; Immunology; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Receptors, Antigen, B-Cell - genetics; Receptors, Antigen, B-Cell - immunology; selection
• ISSN: 1664-3224; 1664-3224
• DOI: 10.3389/fimmu.2024.1473760

The germinal center (GC) reaction is crucial for somatic hypermutation, affinity maturation, and the selection of high-affinity B cells, all of which are hallmarks of the humoral immune response. Understanding the distinct roles of various B cell genes is essential for elucidating the selection mechanisms within the GC reaction. Traditionally, studying B cell gene function in the GC reaction involved generating knock-out mice, a highly time-consuming method that necessitates complex vectors. The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has simplified the creation of knock-out mice. However, even with CRISPR, the generation of knock-out mice still faces challenges, including being time-consuming, costly, having low knock-out efficiency, and raising ethical concerns regarding animal use. To address these challenges, we developed an alternative method to traditional knock-out mouse generation. Our approach entails the CRISPR editing of B cells from transgenic donor mice with different B cell receptor affinities followed by their adoptive transfer into recipient mice. We present a cost-effective, rapid, versatile, and adaptable CRISPR-Cas9 method for loss-of-function studies of individual murine B cell genes within the context of the GC reaction. This method provides a valuable tool for investigating the complex roles of different B cell genes in the GC selection process. As proof of concept, we validated our approach by examining the role of the pro-apoptotic gene Fas in the GC selection process. We adoptively transferred a mix of Fas knock-out (Fas ) low-affinity B cells, Fas wild-type (Fas ) low-affinity B cells, and Fas high-affinity B cells into recipient mice. From our results, Fas low-affinity B cells were still outcompeted by the Fas high-affinity B cells for selection in the GC. An important observation was the accumulation of Fas low-affinity GC B cells when compared to the Fas low-affinity B cells, which suggested a role of Fas in the GC selection process.