Secreted β3-integrin enhances natural killer cell activity against acute myeloid leukemia cells

Abstract

Integrins are a large family of heterodimeric proteins that are involved in cell adhesion, migration, and proliferation. Integrin diversity and function is regulated by alternative splicing. Membrane-bound and truncated β3-integrins were shown to be key players in cancer metastasis. However, the immunomodulatory functions of the soluble (s) β3-integrin have not been investigated yet. In this study, we described a novel form of sβ3-integrin in acute myeloid leukaemia (AML) patients. Furthermore, we assessed the role of the sβ3-integrin in the modulation of natural killer (NK)-cell activity. Levels of sβ3-integrin were analysed in plasma samples of 23 AML patients and 26 healthy donors by ELISA. The capacity of sβ3-integrin to regulate NK cell activity was investigated using proliferation, cytokine secretion, and cytotoxicity assays. Circulating sβ3-integrin was detected in the plasma of 8 AML patients. NK cells showed significantly higher proliferation rates after stimulation with sβ3-integrin and IL-2, IL-15 (73%). Significant increases in the NK cells' secreted levels of TNF-α, IFN-γ were measured in presence of sβ3-integrin. In addition, sβ3-integrin caused the upregulation of Granzyme B transcripts levels as well as FasL expression levels in NK cells. Most importantly, significantly higher K562 or AML blast target cell lysis rates were observed when NK cells were exposed to sβ3-integrin. This study reports the identification of a novel sβ3-integrin in AML patients and provides novel insights into its role in the immunomodulation of NK cell activity.

Figure 1. Detection of soluble β_3-integrin in plasma of AML patients.

Graph display sβ_3-integrin optical density (ODs) in plasma obtained from normal donors (n = 26), non- acute myeloid leukemia (AML) patients (n = 4), and AML patients (n = 23) as obtained by ELISA. The dotted line represents the cut-off value 0.23 units of OD. Levels of significance were expressed as p-values (*p<0.05, **p<0.01, ***p<0.001).

Figure 2. Characterization of sβ_3-integrin detected in AML patient.

(A) Total RNA was prepared from peripheral blood mononuclear cells (PBMCs) of leukemic patients and was reverse transcribed in cDNA. The sβ_3-integrin sequence was amplified by PCR using specific primers. sβ_3-integrin sequences (1201 bp) were detectable in AML patients. Negative controls were also used as described in Materials and Methods. (B) Primer sequences used in the amplification and sequencing of truncated β_3-integrin. Primers used in the sequencing reactions are highlighted.

Figure 3. Soluble β₃-integrin binds natural killer (NK) cells and induces proliferation.

(A) NK cells were cultured without (upper picture) or with the His-tagged sβ₃-integrin (lower picture). Protein binding was detected by fluorescence microscopy upon NK cell staining with a phycoerythrin-conjugated anti-His antibody. Diamidino phenylindol (DAPI) was used to stain the nucleus. Bar scale represents 25 µm. (B) Carboxyfluorescein succinmidyl ester (CFSE)-labelled NK cells were cultured in presence or absence of sβ₃-integrin for 7 days. Figures represent the mean and standard deviation (mean±SD) of cell proliferation percentages of four independent experiments. Levels of significance were expressed as p-values (*p<0.05, **p<0.01, ***p<0.001).

Figure 4. Soluble β₃-integrin increases the cytotoxic potential of NK cells.

Freshly isolated NK cells were cultured in presence or absence of 5µg/ml sβ₃-integrin alone or in combination with IL-2 for 48 h. Non-stimulated NK cells or exposed to a control protein were used as controls. Granzyme B transcript levels were analysed by real time PCR. Housekeeping gene β-actin was used for normalization of cDNA levels. Figure depicts the mean and standard deviations (mean±SD) of values obtained in four separate experiments. Levels of significance were expressed as p-values (*p<0.05, **p<0.01, ***p<0.001).

Figure 5. Expression of FasL is significantly upregulated upon sβ₃-integrin exposure.

Expression of FasL (CD95L) was detected on NK cells previously stimulated with 5 µg/ml sβ₃-integrin alone or in combination with IL-2 for 48 h. Non-stimulated NK cells were also used as control. FasL expression levels were assessed by flow cytometric analyses. (A) Representative dotplots of FasL expression levels upon NK cell culture in the conditions described in Material and Methods. (B) The figure represents the mean and standard deviation (mean±SD) of FasL expression levels detected in four independent experiments. Levels of significance were expressed as p-values (*p<0.05, **p<0.01, ***p<0.001).

Figure 6. Soluble β₃-integrin increases NK cell cytotoxic activity against K562 leukemia cells.

Cytotoxic assays using NK cells previously cultured in the presence or absence of sβ₃-integrin alone or in combination with IL-2 were exposed to primary K562 cells for 6 h at 5∶1 (effector:target) ratio. Target cell lysis was detected by flow cytometric analysis upon 7 aminoactinomycin (7-AAD) staining. (A) The figure shows the mean and standard deviation (mean±SD) of K562 cell lysis detected in four separated experiments. Levels of significance were expressed as p-values (*p<0.05, **p<0.01, ***p<0.001).

Figure 7. Soluble β₃-integrin specifically increases NK cell cytotoxic activity against primary AML blasts.

Cytotoxic assays using NK cells cultured in presence or absence of sβ₃-integrin alone or in combination with IL-2 and a blocking antibody were exposed to primary AML blasts for 6 h at 5∶1 (effector:target) ratio. Target cell lysis was detected by flow cytometric analysis upon 7-AAD staining. The graph depicts NK cell lyse frequencies of AML blasts derived from three leukemic patients.