By developing the present project we have successfully uncovered several key aspects regarding the pro-tumoral role that leukemic ME-sEV (LME-sEV) have in CLL in vivo, deciphering the mechanisms underlying their negative function in cancer. We described that LME-EVs carry immune-checkpoint proteins, such as PD-L1, GAL9, B7-H2, and VISTA. Single-sEV analysis combined with hierarchical stochastic neighbor embedding clustering confirmed that PD-L1 and GAL9 are often coexpressed on CD20+ MHC-II+ vesicles, suggesting LME-sEVs may act as functional units with immunosuppressive capabilities. Using fluorescence-labeled LME-sEVs, we detected that 5% to 10% of the T cells and 40% of B cells uptake sEVs in vivo, and that CD8+ T cells, but not Treg, CD4+ Tconv, and CD19+ B cells, have decreased expression of genes associated with immune response and amino acid transport and increased expression of genes involved in the inhibition of immunity and T-cell differentiation. We have further profiled gene expression and protein contents of sorted CD8+ T lymphocytes treated with LME-sEVs. The genes involved in CD8+ T-cell activation, survival, proliferation, and immune activation are significantly downregulated, whereas genes negatively associated with the above processes were increased in these cells. In addition, LME-sEV treatment inhibited oxidative phosphorylation and leads to an unfavorable metabolic profile in CD8 T cells.
On another hand, we have described the different metabolites that are found inside LME-sEV, proven the intrinsic capacity of sEV to internalise glucose and furthered into the pipelines for identifying the molecules involved in the uptake of sEV by targeted cells. Finally, we have accumulated experience in the development of biologically-engineered sEV for potential therapeutic applications.
