Morfos, V. | Search bioRxiv

bioRxiv2026

Knockout and re-expression system for mutant analysis in primary mouse T cells

We describe here an efficient method for gene editing in mouse T cells, based on well-established, high-efficiency retroviral transduction protocols. Our platform allows analysis of mutant phenotypes in primary murine T cells in vitro and in vivo. This approach uses a single retroviral vector to simultaneously knockout an endogenous gene and ectopically express its mutant version. This knockout/re-expression vector can be used as the only plasmid to transduce Cas9-expressing T cells, or used together with a Cas9 retroviral vector to transduce T cells from any mouse strain. We validated the system for analysis of murine T cells by targeting key molecules in proximal T cell signaling, i.e. CD3{gamma} and Zap70. We obtain high knockout and re-expression efficiencies in both Cas9-expressing and non-Cas9 T cells. Knockout efficiencies can be further improved by gRNA multiplexing. Endogenous proteins compete with their ectopically expressed mutants or tagged versions for cellular location, protein interactions and cellular functions. Here, we quantified the incorporation of CD3{gamma}-GFP into surface T cell receptor (TCR) complexes. Our data shows that the knockout and re-expression platform improves integration of CD3{gamma}-GFP into the TCR. Therefore, eliminating competition between endogenous and ectopic proteins benefits analyses of protein assemblies and signaling pathways in primary T cells. Furthermore, we validated advantages of our system for mutant analysis using wild-type and mutant Zap70s. Zap70 mutants deficient in TCR binding or kinase activity show their phenotypes only in the absence of endogenous protein, further validating our knockout/re-expression approach. Most importantly, this system can be used to generate gene-edited primary T cells for in vivo studies, such as the quantification of anti-tumor responses. Our knockout and re-expression platform provides a useful gene editing tool for primary T cells in fundamental research and immunotherapy development.

immunology↗

bioRxiv2022

Integrated signaling and transcriptome analysis reveals Src-family kinase individualities and novel pathways controlled by their constitutive activity

The Src family kinases (SFKs) Lck and Lyn are crucial for lymphocyte development and function. Albeit tissue-restricted expression patterns, the two kinases share common functions, the most pronounced one, being the phosphorylation of ITAM motifs in the cytoplasmic tails of antigenic receptors. Lck is predominantly expressed in T-lymphocytes; however, it can be ectopically found in B-1 cell subsets and numerous pathologies including acute and chronic B-cell leukemias. The exact impact of Lck on the B-cell signaling apparatus remains enigmatic and is followed by the long-lasting open question of mechanisms granting selectivity amongst SFK members. In this work we sought to investigate the mechanistic basis of ectopic Lck function in B-cells and compare it to events elicited by the predominant B-cell SFK, Lyn. Our results reveal substrate promiscuity displayed by the two SFKs, which however, is buffered by their differential susceptibility towards regulatory mechanisms, revealing a so far unappreciated aspect of SFK member-specific fine-tuning. Furthermore, we show that Lck- and Lyn-generated signals suffice to induce transcriptome alterations, reminiscent of B-cell activation, in the absence of receptor/co-receptor engagement. Finally, our analyses revealed a yet unrecognized role of SFKs in tipping the balance of cellular stress responses, by promoting the onset of ER-phagy, an as yet completely uncharacterized process in B-lymphocytes. SignificanceThe Src-family-kinases Lck and Lyn are mandatory for lymphocyte function. However, several aspects of their regulation and critical pathways they control remain elusive. Using signaling and transcriptome analysis we show that the two kinases share substrate preferences; yet they display differential susceptibility towards regulatory mechanisms, revealing a so far unappreciated aspect of SFK member-specific fine-tuning. Furthermore, overexpression of both kinases suffices to induce receptor-ligation independent signaling responses. Finally, our analyses reveal a novel role of SFKs in tipping the balance of cellular stress responses, by promoting ER-phagy, in the expense of proteasomal degradation and the Unfolded Protein Response. These data advance our understanding of molecular individualities amongst SFK members, and identifies novel networks significant for lymphocyte activation and effector function.

immunology↗