Toll-like receptors (TLRs) play a crucial role in the innate immune response. Although endosomal TLR7 recognizes single-stranded RNAs, their endogenous RNA ligands have not been fully explored. Here, we report 5 '-tRNA half molecules as abundant activators of TLR7. Mycobacterial infection and accompanying surface TLR activation up-regulate the expression of 5 '-tRNA half molecules in human monocyte-derived macrophages (HMDMs). The abundant accumulation of 5 '-tRNA halves also occur in HMDM-secreted extracellular vehicles (EVs); the abundance of EV-5 '-tRNA(HisGUG) half molecules is >200-fold higher than that of the most abundant EV-microRNA (miRNA). Sequence identification of the 5 '-tRNA halves using cP-RNA-seq revealed abundant and selective packaging of specific 5 '-tRNA half species into EVs. The EV-5 '-tRNA(HisGUG) half was experimentally demonstrated to be delivered into endosomes in recipient cells and to activate endosomal TLR7. Up-regulation of the 5 '-tRNA half molecules was also observed in the plasma of patients infected with Mycobacterium tuberculosis. These results unveil a novel tRNA-engaged pathway in the innate immune response and assign the role of "immune activators" to 5 '-tRNA half molecules.
