Membrane Associated TGF-β1 on Leukemia Blast-Derived Microvesicles In Sera of Acute Myeloid Leukemia Patients Suppresses NK Cell Function

Abstract 502

Natural killer (NK) cell cytotoxicity in patients with acute myeloid leukemia (AML) is significantly decreased relative to that in normal controls (NC). TGF-β1 is a potent inhibitor of NK cell cytotoxicity. We considered the possibility that suppression of NK cell activity in AML patients is mediated by leukemia blast-derived microvesicles (MV) via TGF-β1. Sera of 19 patients newly diagnosed with AML prior to any treatment were used to isolate MV by ultracentrifugation. MV from AML patients were positive for the blast-associated markers (CD33, CD34, CD117) by flow cytometry and for membrane-associated TGFb-1 in Western blots, whereas neither these markers nor TGFb-1 were detected in MV from NC. The protein content of blast-derived MV was higher (p<0.001) than that of MV isolated from sera of 25 NC (75μ g±12/mL vs 1.2μ g±0.4/mL). To evaluate MV-mediated NK cell suppression, NK cells obtained from NC were co-incubated with MV isolated from patients' sera. A significant decrease (p<0.002) in NK cell cytotoxicity (2412 LU pre- vs 1640 LU post co-incubation) was accompanied by a concomitant decrease (p<0.004) in the NK-cell activating receptor, NKG2DR, expression levels. To determine whether MV-associated TGF-β1 was responsible for these adverse effects of MV on NK cells, neutralizing anti-TGF-β1 mAb was added to the co-cultures. This mAb significantly abrogated MV-mediated inhibition of NK cell cytotoxicity and down-regulation of the NKG2DR expression level. We next measured the plasma concentrations of TGF-β1 in AML patients and NC. The mean ± SD plasma concentration of TGF-β1 was 2,600 pg/mL ± 2,500 in patients vs. 101 pg/mL ± 55 in NC (p<0.0001). The use of urea, which disassociated membrane-bound TGF-β1 in MV, resulted in a significant increase of the measured TGF-β1 levels (mean 13,700 pg/mL ± 9,800) only in the sera of AML patients but not in the sera of NC. To determine whether the SMAD pathway used by the TGF-β family members is involved in the MV-NK cell interactions, we co-incubated NK cells with MV isolated from sera of AML patients. These MV induced SMAD phosphorylation in NK cells. The addition of anti-TGF-β1 mAbs to the co-culture inhibited SMAD phosphorylation but did not alter expression of the SMAD protein itself. These data provide evidence for a novel mechanism responsible for down-regulation of NK cell activity and NKG2DR expression levels by TGF-β1 associated with leukemia blast-derived MV in AML patients.