CLL Cell-Derived Exosomes Impair Immune Function and Normal Hematopoiesis in CLL

CLL is characterized by immune and hematopoietic abnormalities such as T-cell dysfunction, hypogammaglobulinemia, increased levels of inflammatory cytokines, aberrant monocyte polarity, anemia, neutropenia, and low-grade bone marrow fibrosis. As a result, CLL patients become susceptible to increased rates of infection and/or second neoplasms. Exosomes are nanovesicles that participate in bidirectional communication between neoplastic cells and their microenvironment. Using electron microscopy, we found that CLL cells produce and release CD19 +/CD5 +exosomes, and in agreement with other investigators, we detected high levels of CLL cell-derived exosomes in the plasma of patients with CLL. By using bead-assisted flow cytometry, we found that at least 50% of circulating extracellular vesicles are CLL cell-derived exosomes, identified as CD63 +/CD41a - particles co-expressing CD19 +/CD5 +, whereas only a small proportion of CLL patients' plasma-derived exosomes expressed T-cell or endothelial cell surface markers. Furthermore, we found a positive correlation between levels of CLL cell-derived exosomes in the plasma of patients with CLL and the patients' peripheral white blood cell counts, indicating that CLL cells are the primary source of circulating exosomes. To investigate the effects of CLL cell-derived exosomes, we conducted an analysis of CLL cells and CLL cell-derived exosomes from a cohort of 78 previously untreated patients. To identify the molecular profiles of CLL cell-derived exosomes, we analyzed their RNA transcripts using RNA-seq and qRT-PCR. Our data revealed that CLL cell-derived exosomes expressed significant levels of oncogenic micro-RNAs. Additionally, we identified 1052 differentially expressed genes, and enrichment analysis further unveiled the significant upregulation of gene clusters associated with p53, NF-κB, MAPK, mTOR, and interleukin signal transduction pathways, consistent with their neoplastic cell origin. The distinctive transcriptional profile of CLL cell-derived exosomes led us to explore their effects on normal cell populations. We labeled ultra-centrifuged CLL cell-derived exosomes with fluorescent markers and incubated them with various cell types in culture conditions conducive to their physiological activity. Using confocal microscopy and flow cytometry, we observed significant uptake of CLL cell-derived exosomes by normal monocytes, fibrocytes, B cells, T cells, and CLL cells. Furthermore, incubation with CLL exosomes re