Fig. 2

Principles of the ini-DC/de-ini-DC screening system. Chemical compounds are screened using iniDC differentiated into immature de-iniDC upon withdrawal of dexamethasone (DEX) and doxycycline (DOX). De-iniDC are pulsed with chicken ovalbumin before coculture with B3Z T cell hybridoma cells in a sort of miniature immune system. TCR engagement by B3Z cells results in the production of interleukin-2 (IL2) that can be measured by means of an enzyme-linked immunosorbent assay (ELISA). The genome is screened by using a pooled and barcoded guidance RNA (gRNA) library together with iniDC that stably express the CRISPR-CAS9 nuclease. Upon antigen exposure mature antigen-presenting cells are enriched by immunostaining and flow cytometry. Selected cells are further subjected to next generation sequencing for the identification of gRNAs that induce a gain-of-function phenotype. Single CRISPR RNA gene-edited cells are cloned, differentiated and then employed for DC immunotherapy in vivo. (Created with BioRender.com)