Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood by gradient centrifugation (Lymphoprep, 1.077 g/ml, Nycomed Pharma AS, Oslo, Norway) at room temperature within a few hours after the blood was obtained from the patient.

PBMCs were cryopreserved in 90% heat-inactivated fetal calf serum (FCS, Gibco, Life Technologies, Naerum, Denmark) and 10% dimethyl sulfoxide (Sigma-Aldrich, Broendby, Denmark) with 5–30 × 10 cells/ampoule and stored at −150 °C.

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However, the in vivo impact of 5-Azacytidine on the NK-cell population has to our knowledge never been investigated.

Moreover, effects of 5-Azacytidine on the immune regulatory myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) were investigated as these are key factors inhibiting antitumor immunity.

All analyses were conducted on cryopreserved material.

Manual cell counting was done using trypan blue (Sigma-Aldrich) staining.

Peripheral blood (50 ml) was collected twice during each cycle and processed for later analysis.

Patient characteristics are given in Table 1, and further Material and Methods details according to the MIATA (Minimal Information About T-cell Assays) guideline are provided in Supplementary Table S1.The immunological impact of 5-Azacytidine was evaluated on a diverse cohort of MDS, AML and CMML patients.Peripheral blood was collected and analyzed before and throughout therapy.Furthermore, we analyzed whether single-therapy treatment with 5-Azacytidine induced T-cell responses against CTA-derived epitopes, as previously observed in combination with histone deacetylase inhibition extent the diversity of previously observed responses.Treatment with the demethylating agent 5-Azacytidine leads to prolonged survival for patients with myelodysplastic syndrome, and the demethylation induces upregulation of cancer-testis antigens.