Targeting T cell plasticity by pooled single cell CRISPR-screening in preclinical models of kidney and gut inflammation

Treatment of autoimmune diseases demands a shift from unspecific immunosuppression towards targeted therapies. This could be achieved by turning pro-inflammatory T helper (Th) cells into anti-inflammatory subsets. However, the molecular pathways involved in T cell plasticity and stability are not fully understood. Single cell CRISPR-screens are a powerful tool to simultaneously analyze the impact of multiple genes on cellular phenotypes. To investigate the molecules involved in T cell plasticity in disease settings, we established in vivo single cell CRISPR droplet sequencing (iCROP-seq). By applying this technique to in vivo models of inflammatory diseases in the kidney and intestine, we demonstrate that CRISPR-induced alterations in T cell polarization can be identified and ranked according to corresponding transcriptional perturbations. In particular, we targeted pro-inflammatory Th17 cells in models of immune-mediated diseases and quantified polarization biases into Th1 and regulatory T cells. These findings uncover Th17 to Th1 cell plasticity in the human kidney in the context of renal autoimmunity. iCROP-seq will facilitate the identification of therapeutic targets by highly efficient functional stratification of genes and pathways in a disease- and tissue-specific manner. Processed data file from the alignment with the cellranger software can also be found at Figshare (https://doi.org/10.6084/m9.figshare.28547528). Splenocytes were obtained from Il17a-Cre x Cas9-GFP mice and enriched for CD4+ T cells. With these cells a Th17 polarization was performed. After polarization the cells were transduced with lentiviral particles containing vectors for guide-RNAs and blue fluorescent protein (eBFP2). Transduced cells were then injected in Rag-1-deficient mice. 3 days after cell transfer crescentic glomerulonephritis was induced by i.p. injection of sheep serum against the glomerular basement membrane. Lymphozytes from the kidney were isolated and analysed using scRNAseq. In the disease model of experimental colitis, 14 days prior the injection of the transducted cells into Rag-1-deficient mice, the mice were gavaged with colitogenic bacteria. Intestinal lymphozytes were then isolated and analysed using scRNAseq.