Figure 3 from Extracellular Vesicle Secretion by Leukemia Cells In Vivo Promotes CLL Progression by Hampering Antitumor T-cell Responses

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LME-sEVs enter different lymphocyte subsets and modify CD8+ T cells in the microenvironment. A, Percentage of splenocytes internalizing MB488+ sEVs. Splenocytes from C57BL/6 mice were incubated for increasing periods of time with MB488+ LME-sEVs and analyzed by FC. sEV preincubation with heparin sulfate (Hep) was performed for 4 hours. B, Representative confocal microscopy pictures of total splenocytes after 4 hours of treatment with LME-sEVs, in the absence or presence of heparin (scale bar, 5 μm). C, Splenocytes from C57BL/6 mice were incubated for 24 hours with MB488+ LME-sEVs and then analyzed by FC with lymphocyte-lineage markers. D, FACS-sorted CD4+ and CD8+ T cells were incubated for increasing periods of time with MB488+ LME-sEVs and analyzed by FC. E, Representative confocal microscopy pictures of FACS-sorted CD4+ Tconv cells, CD8+ T cells, and Tregs after treatment with LME-sEVs (24 hours; scale bar, 5 μm). F–H, MB570+-LME-sEVs were i.v. injected in C57BL/6 mice. Total splenocytes were harvested 24 hours later and analyzed by FC directly (F) or after staining for specific immune subsets (CD19+ B cells, CD4+ and CD8+ T cells, G–H). I, Volcano plot showing differential expression of genes (DEG) with FDR <0.05 and log2FC >1 in CD8+ T cells isolated from spleens of mice treated with LME- or HCME-sEVs for 1 week. J, Hierarchical clustering of DEG from I. K, t-distributed stochastic neighbor embedding (t-SNE) of samples from I. L, Hierarchical clustering of selected genes from J, grouped by enriched gene ontologies. M and N, mRNA (M) or protein levels (N) of 3 selected DEG from I, quantified by RT-qPCR or FC, in CD8+ T cells treated in vitro for 48 hours with HCME- or LME-sEVs. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (unpaired Student t test). Data are mean.

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