Background Antileukemic responses of immune reactive cells in AML-patients need to be improved. Combinations of blast-modulatory kitM (GM-CSF+PGE1) (vs control) convert myeloid blasts into dendritic cells of leukemic origin (DCleu), that effectively activate immune-cells against leukemic blasts. Exosomes are small (30–150 nm) membranous vesicles of endocytic origin produced by all cells under physiological and pathological conditions. Their involvement in nearly all aspects of malignant transformation has generated much interest in their biology, mechanisms responsible for information transfer and their role in immune-surveillance as well as -escape.Exosomes secreted by dendritic cells (DCs) have been shown to allow efficient activation of T lymphocytes, displaying potential as promoters of adaptive immune responses.
Materials and Methods 1)DC/DCleu-culture of blast containing AML patients’ whole blood (WB) (n=10) and of healthy volunteers(n=8) with kits, T-cell enriched mixed lymphocyte culture (MLC) with kit- vs un-treated WB, functional blast-cytotoxicity and, leukemia-specificity assays (Degranulation/intracellular cytokine-assays), Flowcytometric evaluation of blast-,DC- and lymphocyte composition before or after cultures. 2)Exosomes were isolated by immunoaffinity from serum, DC- and MLC-culture supernatants of 3 AML patients and 3 healthy volunteers. Exosomes were negatively stained and characterized by transmission electron microscopy (TEM). Fluorescence nanoparticle tracking analysis (fNTA) was performed to determine exosomal size and -concentration. Obtained results were compared in AML and healthy volunteers.
Results Addition of kitM to blast-containing WB significantly increased frequencies of mature DC/DCleu and their subtypes compared to untreated WB without induction of blasts’ proliferation. Immune monitoring showed a continuous increase ofactivated/proliferating cells of the adaptive and innate immune system after Tcell-enriched MLC using kitM pretreated vs -untreated WB, suggesting a production/activation of (potentially leukemia-specific) cells after kit-stimulation. Moreover kit-pretreated WB regularly and significantly improved provision, activation as well as antileukemic and leukemia-specifically directed immune reactive cells after MLC. TEM showed exosome-like structures with a typically cup-shaped appearance without any differences between healthy and AML samples. fNTA revealed average vesicle sizes of 177±23 nm (healthy) and 178±17 nm (AML). Higher levels of EVs were detectable in AML samples compared to healthy controls in serum and after DC-culture, but lower levels after MLC independent of culture conditions.Interestingly, the number of EVs increased during cultivation of DC of AML and healthy samples, but not in AML-derived MLC samples.
Conclusions We will provide data in AML patients and healthy volunteers about a potential role of DCs- and MLC-derived exosomes as biomarkers in immune responses, malignant progression or as potential therapeutic targets for AML patients.
Disclosure Information L. li: None. V. Mussack: None. E. Pepeldjiyska: None. A. Hartz: None. A. Rank: None. C. Schmid: None. E. Özkaya: None. S. Ugur: None. M. Pfaffl: None. H. Schmetzer: None.
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