Patients’ characteristics and diagnostics
According to the Helsinki protocol and the local ‘Ethic Committee’ (Pettenkoferstr. 8a, 80336 Munich, Ludwigs-Maximilians-University Hospital in Munich; Vote-No 339-05), heparinized peripheral whole blood (WB) from healthy (H) donors (n=9) and AML patients (n=9) (Table 1) were collected from the University hospitals of Munich, Stuttgart, Oldenburg and Augsburg after approval by patients’ written informed consent. The cellular composition of AML patients was presented with an average of 50.78% (immune cytologically) IC detected blasts (range 16-82) %. Frequencies of CD3+ cells, CD19+ cells, CD56+ cells and CD14+ cells in AML or H samples are given in Table 2: in case of aberrant CD56, CD19 or CD14 expression on blasts, these data were excluded from NK/B cell or monocyte quantification.
Cellular characterization by flow cytometry
Flow cytometric analyses were evaluated and quantified to acquire the frequencies, phenotypes and subsets of leukemic blasts, DCs, monocytes, B-, T-, NK- and CIK- cells as described before 34. Abbreviations of cell-subsets are given in Table 2a.
Dendritic cell (DC) culture
DC/DCleu were generated from healthy monocytes or myeloid blasts in AML. To stimulate the blasts’ differentiation in WB into DCleu, Kit M (vs. control) was added on day 0 and again after 2-3 days of incubation (800 U/mL GM-CSF (Sanofi-Aventis, Frankfurt, Germany) to induce myeloid differentiation and 1µg/mL PGE1 (PeproTech, Berlin, Germany) for danger signaling and DC-maturation (Table 2b). Cells were harvested after 7-8 days and quantified by flow cytometry as described before 10,34,35.
Mixed lymphocyte culture (MLC)
Immune reactive cells (in WB) were enriched with 1x106 positively selected CD3+ T-cells from healthy or AML samples and co-cultured with a stimulator cell suspension containing 2.5x105 DC/DCleu generated with Kit M (further referred to as MLCWB-DC(M)) in the presence of 50U/mL IL-2 (PeproTech, Berlin, Germany). The same setting without Kit M, served as a control (MLCWB-DC(Control)). Cells were harvested after 7-8 days and used for cytotoxicity fluorolysis assay and for degranulation and intracellular cytokine assays as well as quantified by flow cytometry as described before 9–13,35.
Degranulation Assay (DEG) and Intracellular cytokine assay (ICS)
Cells were mixed and incubated in parallel with vs. without leukemia-associated-antigens (LAA) (PepTivator WT1) and (PepTivator PRAME) for AML samples and with staphylococcal enterotoxin B (SEB) for healthy samples as described9,13,36 The DEG was used to analyze the degranulation activity defined by the surface expression of lysosome associated membrane glycoprotein 1 (LAMP-1, CD107a) of different lymphocyte subsets 9. The ICS was used to analyze the intracellular production of IFNy in different lymphocyte subsets9,11. Abbreviations of cell subtypes are given in Table 2a.
Cytotoxicity Fluorolysis Assay (CTX)
The cytotoxic activity was performed to analyze the lytic activity of Kit M-stimulated (vs unstimulated) T-cell enriched effector cells after 3h and 24h of incubation as described before8,9,34. Cells were analyzed with a FACS Calibur Flow Cytometer and Cell-Quest software (Becton Dickinson, Heidelberg, Germany).
Isolation of EVs from culture supernatants after centrifugation
Before and after DC/MLC different cell-subsets were collected, around 6 ml DC/MLC culture supernatants (DCS/MLCS) of cultivated cells Kit M pretreated vs untreated from each healthy or AML sample were analyzed 37,38 . WB from each healthy or AML sample before treatment with Kit M (H DC/MLC D0 Kit M, AML DC/MLC D0 Kit M), without Kit M as control (H DC/MLC D0 control, AML DC/MLC D0 control) were centrifugated. WB from each healthy or AML sample after treatment with Kit M (H DC/MLC DE Kit M, AML DC/MLC DE Kit M), without Kit M as control (H DC/MLC DE control, AML DC/MLC DE control) were centrifugated.
Cells were sedimented and EVs from DCS/MLCS were prepared after two steps of centrifugation (480 x g, 5 min, 4 °C; then 2,000 x g, 10 min, 4 °C). The DCS/MLCS with EVs were aliquoted in 0.5ml tubes and stored at -80 °C. According to the protocal, 1.27 (± 0.09) ml as input volume were used for EV isolation by Exosome Isolation Kit, pan, human (Miltenyi Biotec) with a further centrifugation step at 10,000 x g for 45 min 39.
Characterization of EVs by TEM and fNTA
As recommended by MISEV2018 guidelines, we characterized some of our EV preparations by TEM and fNTA to assess vesicle morphology, concentration and size distribution as already described elsewhere 40,41. The principle of fNTA, based on the Brownian motion of particles, is not specific for EVs per se. as reported by Mussack et al 39 and a recalculation according to Eitan et al 42 was applied for EVs dilution and volume.
Characterization of EVs by MBFCM
DCS and MLCS were subjected to bead-based multiplex EV analysis by flow cytometry (MBFCM; MACSPlex Exosome Kit, human, Miltenyi Biotec). After centrifugation of thawed culture supernatants at 2,500 x g for 15 minutes, MACSPlex Exosome Capture Beads (containing 39 different antibody-coated bead subsets) were added to each well and counterstained with APC-labelled pan-tetraspanin antibodies (CD9, CD63, CD81) and various antigens on leukocytes, T cell, B cell, monocyte, thrombocyte, integrins, endothelial, or MHC-associated associated antigens as described previously31 (MBFCM antibodies details are given in 41 ).
Statistical analysis
Data are depicted as mean ± standard deviation (SD). A paired t-test was used for comparison of two groups, one-way analysis of variance (ANOVA) with Benjamini-Hochberg adjustments for multiple comparisons was used for more than two groups. Differences were considered as statistically significant (*) with (adjusted) p-values between 0.01 and 0.05. More details are given in 41.