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Extracellular vesicles released by CD40/IL-4 stimulated chronic lymphocytic leukemia cells confer altered functional properties to CD4+ T cells

Dawn T. Smallwood, Benedetta Apollonio, Shaun Willimott, Larissa Lezina, Afaf Alharthi, Ashley R. Ambrose, Giulia De Rossi, Alan G. Ramsay and Simon D. Wagner

Key points

  • CD40/IL-4 stimulated CLL cells release extracellular vesicles (EVs) enriched with specific miRNAs including miR-363.

  • Transfer of CLL-EVs to autologous CD4+ T cells enhances migration and immune synapse formation interactions with tumor cells.

Abstract

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 pseudo-follicles 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 (miR) profiling of EVs released from CLL cells stimulated with CD40 and 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 to 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.

  • Submitted November 16, 2015.
  • Accepted April 20, 2016.