Characterization of Microvesicles in B-Cell Chronic Lymphocytic Leukemia (CLL): A Potential Mediator in CLL B Cell Disease Progression?.

While cellular communication and interactive signaling typically relies on multiple mechanisms including secretion of various growth factors, cytokines, bioactive lipids, and adhesion molecules, we have recently focused on cell-to-cell communication that involves circular membrane vesicles designated as microvesicles (MVs). These are normal constituents of blood plasma released by leukocytes, endothelium, platelets and erythrocytes and are generated by cell activation and growth. In normal serum, the order of MV abundance is platelet-derived (80%), endothelial (10%) and leukocyte-derived (10%) but MV can also be released by malignant cells. The size varies from 0.1–1.0 μm and, MVs express antigens characteristic of the cell of origin, carry other membrane and cytoplasmic constituents and exhibit negatively charged phospholipids (phosphatidylserine) detectable by Annexin V binding. To begin to characterize MV and their potential for communication in CLL, we isolated and characterized MVs from the plasma of chronic lymphocytic leukemia B-cell (CLL B) patients (Rai 0-IV). After isolation from platelet-free plasma, MVs were characterized by flow cytometry for forward and side scatter using 1μm fluorescent beads and stained with Annexin V. Our results suggest that MVs are heterogeneous in size with the majority of MVs within 1μm. Transmission electron microscopy confirmed the size heterogeneity of MVs and exhibited the morphology of the membrane-bound vesicles after phosphotungstic acid staining onto parlodium-coated 300 mesh copper grids. Flow cytometric analysis further demonstrated the presence of CD61 (platelet/megakaryocyte marker), CD19, CD5, CD52, and CD20 on the surface of MV, albeit in differential amounts depending on the stage of the disease and the total leukocyte count. For example, in Rai stage IV (n= 5), we found lesser numbers of MV carrying platelet-derived marker CD61 and more CD19 (41–79%), and CD5 compared to normal MV; 1–5% MV from healthy controls expressed CD19 while Rai stage 4 patients MV expressed CD19 from 41–79%. These results suggest that a significant number of MVs are generated from the leukemic B-lymphocytes in more advanced Rai stage. However, we also found that patients with a higher blood lymphocyte count but lower Rai-stage (Rai I/II) contain moderate levels of CD19-bearing MVs (13–43%). Importantly, presence of MVs carrying CD52/CD20 in the blood of CLL patients may undermine the therapeutic effectiveness of alemtuzumab and rituximab. To this end, we observed that MVs could be incorporated into the CLL B cells as evident from the expression of the platelet antigen CD61 on CLL B cell surfaces after co-culture of CD61-bearing MVs with CLL B cells. Global proteomic analysis of MVs isolated from an advanced stage (Rai IV) CLL B patient detected the presence of about 700 proteins that normally reside in the nucleus, cytoplasm or membrane. In summary, we have isolated and characterized plasma MV from CLL B patients and found them to be more likely generated from CLL B cells in relation to disease stage or total lymphocyte count. These MVs contain a multitude of proteins and are able to integrate into bystander CLL B cells. We also found that these MVs carry a large set of proteins, which could potentially not only modulate cell-cell communication but also interrupt monoclonal antibody directed therapy. Further studies are underway to dissect their roles in CLL B malignancy.