Circulating Exosomes Carrying an Immunosuppressive Cargo Interfere with Adoptive Cell Therapy in Acute Myeloid Leukemia

Introduction: Adoptive immunotherapy, including transfer of activated NK cells (aNK), is currently under active investigation for patients with refractory/relapsed acute myeloid leukemia (AML). However, the highly immunosuppressive microenvironment in patients with AML may create a major barrier for adoptive immunotherapy. Exosomes, virus-size (30-100 nm) membrane-bound vesicles found in all body fluids, are produced in abundance by leukemic blasts and are strongly immunosuppressive. The presence of exosomes in the plasma of relapsed/refractory AML patients could impair anti-leukemia activity of adoptively transferred aNK cells. To test this hypothesis, we evaluated ex vivo effects of exosomes isolated from plasma of AML patients on the activity of aNK cells which were used for adoptive immunotherapy in a Phase 1 clinical trial (NCT00900809) for patients with relapsed/refractory AML performed at our institution.

Methods: Venous blood (20-50 mL) was obtained from patients with refractory/relapsed AML (n=7) prior to and after therapy with adoptively-transferred aNK cells. Exosomes were isolated from the patients' plasma as well as normal controls' (NC) plasma by size exclusion chromatography on Sepharose 2B. Exosome numbers and size (qNano), morphology (TEM) and protein levels were determined. The presence of exosome-related markers (Tsg101, CD81), myeloid cell-surface markers associated with AML (CD123, CLL-1, CD44, CD96) and of immunosuppressive proteins (CD39, CD73, PD-1/ PDL-1, Fas/FasL, TGF-β1) in exosomes was studied by Western blots. The AML plasma-derived exosomes were co-incubated with normal human NK cells or aNK cells used for therapy. Up-take of PKH26-labeled exosomes by recipient aNK cells was monitored by confocal microscopy and changes in NKG2D expression levels on aNK cells or in NK-cell cytotoxicity by multiparameter flow cytometry. Exosome-induced signaling via receptors (A_2AR, TGFβR, CD95 or PD-1) on aNK cells was studied by Western blots ± inhibitors of each relevant pathway.

Results: The protein levels of exosome fractions in plasma of refractory/relapsed AML patients prior to adoptive therapy with aNK cells were significantly higher (70 ± 15 μg protein/mL) than those isolated from plasma of NCs (35±2.7 μg protein/mL) (P<0.05). Exosome protein levels remained persistently elevated after therapy with the aNK cells. The molecular profile of exosomes in AML patients' plasma was distinct from that of exosomes in plasma of NCs; in addition to leukemia-associated antigens (LAA), AML exosomes were enriched in TGF-β1 (LAP), CD39 and CD73 ectoenzymes, PD-L1 and FasL. Co-incubation of these AML exosomes with the aNK cells that express cognate surface receptors for the above listed ligands or ectoenzymes down-regulated survival and lytic functions of aNK cells. Pharmacologic inhibitors or blocking anti-PD1 mAbs and anti-TGF-β1 mAbs prevented apoptosis and partly abrogated dysfunction of aNK cells. Inhibitory in vivo effects of the high plasma exosome levels in plasma of AML patients was demonstrated by the absence of evidence for up-regulation of NK cytotoxicity in the peripheral blood of the patients receiving adoptive therapy with aNK cells.

Conclusions: Persistently elevated levels of biologically-active immunosuppressive exosomes carrying leukemia-associated antigens and various immunosuppressive proteins in plasma of patients with relapsed/refractory AML interfere with anti-leukemia activity of adoptively transferred aNK cells. Blast-derived exosomes emerge as a significant deterrent to a success of adoptive immunotherapies with activated immune cells.