Figure 5 from Extracellular Vesicle Secretion by Leukemia Cells In Vivo Promotes CLL Progression by Hampering Antitumor T-cell Responses

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LME-sEVs decrease CD8+ T-cell functions. A and B, Percentages (A) and numbers (B) of CD62L−KLRG1+ CD8+ T cells after 48 hours of treatment with LME- and HCME-sEVs assessed by FC. C, Expression of ICP on CD62L−KLRG1+ CD8+ T cells from B. HSNE clustering depicting treatments, cluster identity, and marker expression. D, Hierarchical clustering based on ICP expression. E, Percentages of PD1+TIM3+ICOS+ CD8+ T cells from cluster C1 (top) and of PD1+LAG3+TIM3+TIGIT+ICOS+ CD8+ T cells from cluster C8 (bottom). F–I, CD8+ T cells were isolated from C57BL/6 and CLL cells from TCL1 mice. F, Percentage of T cell–mediated killing of TCL1 cells (cytotoxic assay) in the presence of HCME-sEVs or LME-sEVs (N = 6). G, Quantification of CD8+ T-cell:TCL1 cell conjugates upon treatment with LME- or HCME-sEVs (N = 3–4) and representative images (scale bar, 10 μm). H, Quantification of immune synapse formation (F-actin area in μm2, HCME-, n = 31 and LME-sEVs, n = 39, dashed line representing median) and representative medial optical sections (scale bar, 5 μm) with arrows indicating the synapse. I, Mean Fluorescence intensity (MFI) of GzmB at the synapse between CD8+ T and CLL cells (HCME-, n = 31 and LME-sEVs, n = 51) and representative 3D volume-rendered images. J, miRNA levels quantified by RT-qPCR in CD8+ T cells treated with HCME- or LME-sEVs for 24 hours. K, Protein levels of miRNA targets determined by FC in CD8+ T cells treated for 48 hours with HCME-sEVs or LME-sEVs transfected with scramble or antagomiRs (miR-150, -155, and -378a). Preincubation of LME-sEVs with heparin was used as an inhibitor of sEV internalization. L, ICP levels determined by FC in CD8+ T cells treated for 48 hours with HCME-sEVs or LME-sEVs preincubated with blocking Abs (PD-L1, GAL9, VISTA, and MHC-II) or corresponding isotypes. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (unpaired Student t test). Data are mean.

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