699 A CRISPR-Cas9 screen identifies negative regulators of CXCL9 expression by dendritic cells
Year: 2026
Type: conference-abstract
Abstract: Background Type I conventional dendritic cells (cDC1s) are critical for initiating and sustaining T cell-mediated anti-tumor immunity. Key molecules involved in this process include the chemokine CXCL9 and the cytokine interleukin-12, which work together to recruit and stimulate T cells, respectively. However, expression of these molecules is often suppressed within tumors, reflecting an opportunity for therapeutic intervention.Methods We performed a targeted and whole genome CRISPR-Cas9 functional genetic screen in vitro with a murine splenic cDC1 cell line to identify regulators of CXCL9 protein expression during interferon-gamma (IFN-g) stimulation. Findings were confirmed with individual knockout lines and target expression was characterized in vivo.Results One of the top negative regulators identified in both screens was Activin A Receptor Like Type I (Acvrl1), a TGF-beta family type-I receptor known as ALK1 that serves as a receptor for bone morphogenic protein (BMP) 9 and 10 in endothelial cells. For validation, ALK1 was knocked-out (KO) in MutuDC1940 cDC1s by CRISPR/Cas9 gene deletion, with ALK1 KO cells expressing 1.5-fold more CXCL9 following IFN-g stimulation (p < 0.0001). Real time quantitative PCR revealed a 2.6-fold increase in CXCL9 mRNA during IFN-g stimulation (p < 0.0001), indicating transcriptional level regulation of CXCL9 by ALK1. Bulk RNA sequencing confirmed changes in CXCL9 expression and revealed additional changes in the expression of IFN-g stimulated genes. Mouse splenic and tumor cDC1s expressed ALK1, with expression also observed on cDC2s and macrophages. A similar expression profile was observed in human breast tumors through an analysis of public single cell RNA sequencing datasets.Conclusions Here we identify ALK1 as a negative regulator of CXCL9 expression by murine cDC1s in vitro, with ALK1 expressed by splenic and tumor cDC1s. Future studies will evaluate the in vivo role of ALK1 expression by cDC1s, explore its potential as a therapeutic target, and ascertain the mechanism by which it regulates CXCL9 gene expression.Ethics Approval All mice were maintained in accordance with Institutional Animal Care and Use Committee (IACUC) standards followed by the Moffitt Cancer Research Center (Tampa, Florida).
Institutions: University of South Florida, Moffitt Cancer Center
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,490
OpenAlex ID: W7225296846