Evidence for the Role of Biologically Active Exosomes in Acute Myeloid Leukemia Relapse

Introduction: Despite of the notable achievements of multi-modality treatments in patients with acute myeloid leukemia (AML), leukemic relapse remains a formidable clinical challenge. Studies in AML patients have shown that tumor-induced impairments of immune cells contribute to leukemia progression. One recently identified immunosuppressive mechanism operating in AML is the release by leukemia blasts of virus-size (30-100nm) membrane-bound vesicles called exosomes. We have reported that plasma of patients with newly-diagnosed AML prior to any therapy contains higher levels of exosomal proteins than that of normal donors. AML exosomes carry an immunosuppressive cargo, including membrane-associated TGF-β1, MICA/MICB and markers of myeloid blasts. We hypothesize that persistence of these exosomes may contribute to the high relapse rates seen in AML.

Methods: Venous blood (20-50 mL) was obtained from patients newly diagnosed with AML prior to any treatment, from AML patients who achieved CR following initial therapy and from age-matched healthy volunteers (NC). Exosome fractions were isolated from plasma by using mini-size exclusion chromatography with Sepharose 2A. Exosome protein levels, numbers and size of exosomes (qNano) and their morphology by transmission electron microscopy were determined. Exosomes were characterized by western blots for expression of exosome markers, Tsg101 and CD81, and myeloid cell-surface markers associated with AML, interleukin-3 receptor alpha chain (CD123) and C-type lectin-like molecule-1 (CLL-1), CD44 and CD96. Isolated normal human NK cells were co-incubated with AML exosomes and multiparameter flow cytometry was used to monitor changes in expression levels (mean fluorescence intensity) of NKG2D on NK cells.

Results: Exosome fractions isolated from AML patients' plasma at diagnosis (n=16) had higher mean protein content than fractions obtained from NC plasma (96 vs 37 µg protein/mL plasma, p=0.03). In exosome fractions isolated from plasma of AML patients in CR (n=13) protein levels remained elevated (mean value=120 µg protein/mL plasma) at the time when leukemic blasts were undetectable in the bone marrow by conventional hematopathological methods. AML exosomes in patients in CR carried blast markers, CD123, CLL-1, CD44, CD96 and were enriched in TGF-β1. Co-incubation of these exosomes with activated NK cells resulted in down-regulation of NKG2D expression with a concomitant reduction of NK-cell cytotoxicity. Ten/13 AML patients in CR whose plasma exosome levels remained elevated eventually relapsed.

Conclusion: The persistently elevated levels of biologically-active TGF-β1+ exosomes carrying leukemia blast markers in plasma of AML patients in CR impair anti-leukemia immune responses and might contribute to leukemia relapse.