CRISPR screenings reveal neuropeptide orchestrate T helper cell fate decision

T helper type 1 (Th1) cells play critical roles in viral infection response and autoimmune disease. Recent studies have implicated the imbalance of T helper subtypes is correlated with outcome of COVID-19 infection, however, the underlying mechanism that balancing Th1 cells versus other T helper cells remains unknown. Here, we profiled the heterogeneity and dynamic regulation during in vivo and in vitro Th1 differentiation using scRNA-seq and defined the Th1 unique program using a Th1/Th2 dichotomous differentiation condition. To further validate the functions of defined Th1 regulators, we performed CRISPR screenings for both in vitro as well as in vivo Th1 differentiation. RAMP3 as one of the receptors for neuropeptide CGRP was defined as a novel regulator balancing Th1 and Th2 differentiation and was proved an indispensable role for Th1 response during virus infection. CGRP, RAMP3 and cAMP signaling form a positive feedback loop to enhance Th1 differentiation through activating STAT1. Together, ur work highlights the network between CGRP-mediated neuronal signaling and immune response during virus infection. Overall design: 1. To explore the dynamic regulation during in vitro T cell differentiation, we performed temporal bulk RNAseq during in vitro T cell differnetiation under Th0, Th1 and Th2 conditions. 2. To explore the gene regulation during in vivo Th1 differentiation, we performed single cell RNA-seq of splenocyte upon LCMV Armstrong infection. 3. To further explore the interaction between Th1 and Th2 regulation networks, we performed tepmoral single cell RNA-seq under Th1-Th2 bifurcation condition. 4. To reveal the effect of CGRP, we performed RNA-seq and ATAC-seq upon CGRP treatment under both Th1 and Th2 polarization conditions.