CLL Exosome-Derived Y RNA hY4 Induces TLR7/8-Mediated Inflammation and PD-L1 Expression in Monocytes

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy associated with an inflammatory milieu and impaired anti-tumor immunity. Both in human CLL samples and the Eµ-TCL1 mouse model of CLL, monocytes and macrophages were identified as key players in the involved processes, as they secrete immune regulatory cytokines and show enhanced expression of the immune checkpoint protein PD-L1 which is known to be involved in T-cell suppression. We recently showed that reactivation of T-cell activity using anti-PD-L1 antibodies controls leukemia development in mice and is associated with a normalization of CLL-associated immune defects.

The current study aimed at unraveling the molecular mechanisms of CLL-induced changes in monocytes and macrophages and focused on CLL-derived exosomes and their role in the tumor microenvironment.

Exosomes were isolated from blood plasma of CLL patients as well as culture supernatant of the CLL cell line MEC-1 by a serial centrifugation protocol. Characterization of isolated exosomes by electron microscopy, Nanoparticle Tracking Analysis (NTA), and Western blot analysis revealed vesicles, 30 to 350 nm in size, which were positive for various exosome marker proteins. Quantification of exosomes by NTA and ELISA indicated an enrichment of B-cell derived exosomes in plasma of CLL patients compared to healthy controls, although absolute exosome counts were not different. RNA sequencing and proteome analysis of CLL exosomes revealed a disease-specific composition and identified non-coding Y RNA hY4 as the most abundant exosomal RNA species. Transfer of CLL exosomes or hY4 RNA alone to monocytes triggered release of cytokines like CCL2, CCL3, CCL4, and IL-6, and increased expression of PD-L1. As these are key features associated with CLL, a novel role for exosomal Y RNAs in the tumor microenvironment of CLL is suggested. Of interest, exosome or hY4-induced responses in monocytes were significantly reduced by chloroquine treatment and completely abolished in TLR7-deficient monocytes. Therefore, exosomal hY4 was identified as novel ligand that activates Toll-like receptor 7/8 signaling pathway in monocytes.

Tumor-derived exosomes as well as exosomal Y RNAs were detected in a number of malignancies, suggesting their more general contribution to cancer-related sterile inflammation and the formation of a tumor-supportive myeloid microenvironment. Ongoing studies will show whether these novel findings can be exploited in treatment approaches for CLL and other malignancies.