Table 1.

Summary of discoveries and protocols used in the main articles about CLL EVs discussed in the review (UC—ultracentrifugation; CLL cell lines: EHEB—HG3—MEC-1—PGA-1; endothelial cell lines: HMEC-1—HUVEC; human BM cell lines: HS5; monocytic cell line: THP-1; primary fibroblasts: HDFn; ↑: increase; ↓: decrease).

Donor Cells	EVs	EV Markers	Cargo	Purification Protocol	Target Cells	Results	Ref
Untreated CLL patients (n = 34) and healthy donors, MEC-1	EXO	TSG101, MHC I/II, IgM, Lyn kinase, CD81, CD37, ITGA4	miR-202-3p	10 min at 500× g, 10 min at 4000× g, 30 min at 18,000× g, filtration (Immuno-magnetic isolation), UC 90 min at 100,000× g (×2)	HS-5	CLL EXOs ↑ expression of genes such as c-fos and ATM; ↑ proliferation of recipient HS-5 cells; CLL EXOs enriched in certain miRNAs	[44]
Primary CLL B-cells and normal B cells, plasma (n = 33 CLL/n = 9 treatment-naïve CLL patients/n = 5 CLL patients under ibrutinib)	MV	CD52	-	20 min at 2500× g (×3); 1 h at 16,000× g at 4 °C	-	↑ CD52+ MVs with BCR stimulation in CLL B-cells; ↑ plasma CD52+ MVs correlated to tumor progression; ↓ plasma CD52+ MVs after ibrutinib therapy	[45]
Primary CLL and healthy B cells	EV	CD63, CD9, CD54, CD82	TCL1A-mRNA	10 min at 300× g, 15 min at 6800× g (×2), UC 90 min at 100,000× g (×2)	HDFn and THP-1	CLL-CpG-EVs contain disease-relevant mRNA; ↑ CLL-EVs compared to healthy B cells	[46]
CD19+ B cells from CLL patients and healthy donors	EXO	CD63, CD9, CD37	miR-155, miR-150	10 min at 300× g, 10 min at 2000× g, 30 min at 10,000× g at 4 °C, UC 70 min at 100,000× g at 4 °C (×2)	-	↑ EXOs in CLL patients’ plasma; ↑ EXOs with BCR activation by α-IgM in CLL B cells; ibrutinib impedes α-IgM-stimulation EXO release; ↑ EXO miR-150 and miR-155 with BCR activation	[47]
Serum (n = 131 CLL/n = 28 healthy controls)	MV	CD19, CD37		20 min at 2000× g at 4 °C, 30 min at 10,000× g at 4 °C, UC 70 min at 100,000× g at 4 °C (×2)	-	↑ MVs in CLL patients’ plasma; CD19+ CD37+ MVs correlate to tumor progression; total MVs predict for overall survival and time to treatment	[48]
Plasma (n = 60 CLL, n = 5 healthy controls)	MV	CD19		20 min at 2500× g (×3), 1 h at 16,000× g at 4 °C	HS-5, primary BMSCs	↑ MVs in CLL patients’ plasma; ↑ VEGF, B-catenin pathway, cyclin D1, and c-myc in CLL-BMSCs	[50]
CD19+ CD5+ B cells CLL patient and healthy donors; plasma CLL patients	EV comprise EXO	-	miR-363, miR-155, miR-374b	5 min at 250× g at 4 °C, 10 min at 2000× g at 4 °C, 30 min at 10,000× g at 4 °C, UC 110 min at 100,000× g	CD4+ T cellsfrom CLL patients	CD40/IL-4–stimulated CLL cells released specific EV miRNAs; ↑ migration; proliferation of CD4+ T cells; immunological synapse signaling	[51]
Primary CLL (n = 21), MEC-1	EXO	ALIX, TSG101, HLA-DR		10 min at 400× g (×2), 20 min at 2000× g, filtration 0.45 μm, UC at 110,000× g at 4 °C, flotation on Optiprep cushion (Axis-Shield, 17%) for 75 min at 100,000× g at 4 °C, filtration 0.45 μm	Human BM-MSCs, HMEC-1, HS5	CLL-EXO transfer protein and miRNA into stromal cells that induce a CAF-like phenotype; uptake by endothelial cells ↑ angiogenesis	[52]
MEC-1	EXO	CD63, CD9	miR-146a	10 min at 400× g (×2), 20 min at 2000× g, filtration 0.45 μm, UC 70 min at 110,000× g (×2), 75 min at 100,000× g at 4 °C, filtration 0.45 μm	Human BM-MSCs	CLL cells deliver miR-146a to BM-MSCs that induce CAFs phenotype by down-regulation of USP16 mRNA expression	[53]
Human BM-MSCs	EV	CD63		10 min at 300× g (×2), concentrated on 3 K centrifugal device, UC 1 h at 150,000× g at 4 °C (×2)	Primary CLL	BM-MSCs ↓ B CLL spontaneous apoptosis and ↑ chemoresistance to fludarabine, ibrutinib, idelalisib and venetoclax; ↑ CLL B cells migration	[54]
Plasma CLL patients, MEC-1	EXO	CD63, CD81, TSG101	CLIC1	20 min at 400× g, 40 min at 2000× g, filtration 0,45 μm, UC 70 min at 110,000× g at 4 °C, floatation on Optiprep cushion (Axis-Shield, 17%) 75 min at 100,000× g at 4 °C, filtration 0.45 μm	HUVECs	MEC-1 EXO invasion; metastasis and angiogenesis of HUVECs by transferring CLIC1	[55]
Plasma CLL patients and healthy donors, MEC-1	EXO	RAB5a, HSP70, HLA-DR, CD81	noncoding Y RNA hY4	300× g and 10,000× g, UC at 100,000× g, UC on 40% sucrose cushion	Human monocytes or murine BM-MDSCs	↑ release of cytokines, such as CCL2, CCL4, and interleukin-6,and expression of PD-L1	[56]
Untreated CLL patients (n = 26, aggressive /indolent)and healthy donors, MEC1 and HG3	EXO	CD63, CD81	NAMPT	15 min at 3000× g, ExoQuick -TC reagent overnight at 4 °C, 30 min at 1500× g	Primary monocytes (CD14+ CD16+)	CLL-EXO transfer NAMPT to monocytes; ↑ NAD+ (nicotinamide adenine dinucleotide) which activatedSIRT1-C/EBPβ signaling pathway in monocytes	[57]
CLL patients (n = 56) and healthy donors, EHEB and MEC1, serum CLL patients and healthy donors	EXO	CD63, CD81		10 min at 500× g, 20 min at 16,500× g, filtration 0.2 μm, UC 70 min at 110,000× g at 4 °C (×2) and filtration 0.2 μm	CD14+HLA-Drlow monocytes (MDSCs)	miR-155 in CLL-EXO induces MDSCs; is disrupted by vitamin D	[58]
PB and plasma samples, CLL patient and healthy donors, MEC-1, HG-3, EHEB, and PGA1	EV	CD9, CD63, CD81, CD19, CD20, CD40	ICs	filtration 0.2 μm, UC 70 min at 110,000× g at 4 °C	T-cells	CLL-EV contain ICs that may hamper T-cell viability, proliferation, activation, and metabolism	[59]
Eμ-TCL1 CLL murine model (WT B cells from C57BL/6 mouse)	sEV (EXO)	Alix, TSG101, CD63, CD9, CD81	miR-150, -155, -21, -146a, -378a, and -27a, IC ligands	5 min at 400× g, 20 min at 400× g, 40 min at 2000× g, 60 min at 10,000× g, filtration 0.2 μm, UC 70 min at 110,000× g at 4 °C, flotation on 17% Optiprep cushion, 75 min at 100,000× g at 4 °C, UC 70 min at 110,000× g at 4 °C, filtration 0.45 μm and 0.22 μm	CD8+ T cells	small EVs secreted by CLL cells in mouse model inhibit CD8+ T-cell immune response against tumor cells	[60]
Serum (n = 9 CLL/n = 18 healthy controls)	EV			-	-	↑ miR-155 in CLL EVs	[61]