Perforin 2 controls unconventional cytokine release from mucosal APC Project Summary How professional antigen presenting cell (APC) populations focally deliver cytokines to T cells for shaping the pro-inflammatory vs. anti-inflammatory status of mucosal tissue remains incompletely understood. In particular, cytokines that lack N-terminal peptide sequence such as the IL-1 family cytokine IL-33 can't access conventional protein secretion pathways, which has led to the prevailing view that cell death is responsible for IL-33 release. This project is built upon an exciting set of preliminary data demonstrating that mucosal conventional dendritic cell (cDC) subsets, in both humans and mice, express the transmembrane pore-forming protein Perforin-2, which at least in mice, facilitates IL-33 secretion. While in human cDC, we find Perforin-2 expression primarily in an interferon regulatory factor 4 (IRF4) subset indicating the cDC2 lineage, in mouse cDC, we find Perforin-2 in the CD103+ cDC1 subset known to express the transcription factors Irf8 and Batf3. Irrespective of this lineage distinction, both human and mouse CD11c+ cells in the respiratory mucosa contain cytoplasmic IL-33 protein. Our data demonstrate that inhibition of Perforin-2 activity prevents IL-33 release from cDC and inhibits the proliferative expansion of a poorly understood ST2+GATA3+Foxp3+Treg subset. Given that Perforin-2 has been shown to also regulate Type 1 IFN signaling through controlling IFNAR responsiveness, we propose an important regulatory mechanism exists in humans and mice that is dependent upon mucosal APC that express Perforin-2. This project tests the central hypothesis that APC require Perforin-2 as an inducible plasma membrane conduit for unconventional cytokine delivery at the mucosal interface. Three specific aims (SA) will be investigated. SA1 will determine whether Perforin-2+ APC predict Treg subset abundance in sinonasal mucosa and define the transcriptional landscape of Perforin-2+ APC. SA2 will define the Perforin-2 domains required for IL-33 release, the diversity of Perforin-2-dependent secreted molecules, and the requisite intracellular trafficking machinery responsible for Perforin-2 plasma membrane localization during APC-T cell interactions. SA3 will directly test whether cDC1 and/or cDC2 subsets preferentially use Perforin-2 for driving pathogen-specific T cell responses in mouse models of respiratory virus or helminth infection. Taken together, this MIST project stands to uncover a new paradigm for understanding how cDC instruct immunity within the respiratory mucosa.