ZEB2 orchestrates a transcriptional program to safeguard the integrity of human CD4+ Th1 EM cells

CD4+ Th1 cells migrate to sites of inflammation, where they are indispensable for eliminating intracellular pathogens. The lineage defining transcription factor T-bet establishes the Th1 transcriptional programme, directing IFNG to drive effector responses and inducing subordinate transcription factors to shape their phenotype. Aberrant Th1 cell activity drives the pathogenesis of multiple autoimmune diseases, but the detailed mechanisms by which Th1 cells maintain or lose their integrity remain largely uncharacterised. Using immunogenomics and high resolution immune phenotyping in human CD4+ cells, we discovered that the transcription factor ZEB2 is lineage restricted to Th1EM cells. Detailed molecular validation using CRISPR/Cas9 deletion of ZEB2, whole genome transcriptomics and pathway mapping has revealed that ZEB2 is a signalling hub for multiple pathways, stabilising the integrity and function of human CD4+ Th1EM cells. Furthermore, our disease linked pathway mapping suggests that ZEB2 is implicated in the control of Th1-mediated autoimmune disease. To investigate the role of ZEB2 in Tconv cells, we utilized CRISPR/Cas9 technology for gene deletion in human Th1 Tconv cells. The procedure began with the resuspension of three sgRNAs targeting ZEB2 and a non-targeting control sgRNA to a final concentration of 100µM, following which the ZEB2-targeting sgRNAs were mixed to achieve a 40µM concentration. Ribonucleoprotein (RNP) complexes, comprising the sgRNAs and 2NLS Cas9 nuclease, were prepared and incubated with Nucleofection™ buffer. Purified Th1 cells (CD4+ CD25lo CD127hi CD45RA-,CXCR3+, CCR6-), previously activated and then washed, were resuspended in Human T Cells Nucleofector™ Solution and electroporated using the Amaxa® Nucleofector® 2b device with a T-020 pulse setting for activated T cells. Post-electroporation, cells were allowed to recover in prewarmed Complete X-VIVO T cell medium supplemented with IL-2 and then cultured in a 24-well plate. Medium replacement occurred 24 hours post-electroporation to facilitate optimal cell recovery, and after 48 hours, viable cells were sorted for Th1 central memory (CD62L+) and Th1 effector memory (CD62L-) and RNA sequencing were performed.