Extracellular vesicles released by CD40/IL-4-stimulated CLL cells confer altered functional properties to CD4+ T cells.

The complex interplay between cancer cells, stromal cells, and immune cells in the tumor microenvironment (TME) regulates tumorigenesis and provides emerging targets for immunotherapies. Crosstalk between CD4(+) T cells and proliferating chronic lymphocytic leukemia (CLL) tumor B cells occurs within lymphoid tissue pseudofollicles, and investigating these interactions is essential to understand both disease pathogenesis and the effects of immunotherapy. Tumor-derived extracellular vesicle (EV) shedding is emerging as an important mode of intercellular communication in the TME. In order to characterize tumor EVs released in response to T-cell-derived TME signals, we performed microRNA (miRNA [miR]) profiling of EVs released from CLL cells stimulated with CD40 and interleukin-4 (IL-4). Our results reveal an enrichment of specific cellular miRNAs including miR-363 within EVs derived from CD40/IL-4-stimulated CLL cells compared with parental cell miRNA content and control EVs from unstimulated CLL cells. We demonstrate that autologous patient CD4(+) T cells internalize CLL-EVs containing miR-363 that targets the immunomodulatory molecule CD69. We further reveal that autologous CD4(+) T cells that are exposed to EVs from CD40/IL-4-stimulated CLL cells exhibit enhanced migration, immunological synapse signaling, and interactions with tumor cells. Knockdown of miR-363 in CLL cells prior to CD40/IL-4 stimulation prevented the ability of CLL-EVs to induce increased synapse signaling and confer altered functional properties to CD4(+) T cells. Taken together, these data reveal a novel role for CLL-EVs in modifying T-cell function that highlights unanticipated complexity of intercellular communication that may have implications for bidirectional CD4(+) T-cell:tumor interactions within the TME.