Integrin beta 7 (ß7), a subunit of the integrin receptor, is expressed on the surface of immune cells and mediates cell–cell adhesions and interactions, e.g., antitumor or autoimmune reactions. Here, we analyzed, whether the stimulation of immune cells by dendritic cells (of leukemic derivation in AML patients or of monocyte derivation in healthy donors) leads to increased/leukemia-specific ß7 expression in immune cells after T-cell-enriched mixed lymphocyte culture—finally leading to improved antileukemic cytotoxicity. Healthy, as well as AML and MDS patients’ whole blood (WB) was treated with Kit-M (granulocyte–macrophage colony-stimulating factor (GM-CSF) + prostaglandin E1 (PGE1)) or Kit-I (GM-CSF + Picibanil) in order to generate DCs (DCleu or monocyte-derived DC), which were then used as stimulator cells in MLC. To quantify antigen/leukemia-specific/antileukemic functionality, a degranulation assay (DEG), an intracellular cytokine assay (INTCYT) and a cytotoxicity fluorolysis assay (CTX) were used. (Leukemia-specific) cell subtypes were quantified via flow cytometry. The Kit treatment of WB (compared to the control) resulted in the generation of DC/DCleu, which induced increased activation of innate and adaptive cells after MLC. Kit-pretreated WB (vs. the control) led to significantly increased frequencies of ß7-expressing T-cells, degranulating and intracellular cytokine-producing ß7-expressing immune cells and, in patients’ samples, increased blast lysis. Positive correlations were found between the Kit-M-mediated improvement of blast lysis (vs. the control) and frequencies of ß7-expressing T-cells. Our findings indicate that DC-based immune therapies might be able to specifically activate the immune system against blasts going along with increased frequencies of (leukemia-specific) ß7-expressing immune cells. Furthermore, ß7 might qualify as a predictor for the efficiency and the success of AML and/or MDS therapies.

Background: Extracellular vesicles (EV) are cell-derived vesicles released by all cells in health and disease. Accordingly, EVs are also released by cells in acute myeloid leukemia (AML), a hematologic malignancy...

were analysed using the CEL-seq2 platform. Interactions between tumour and immune cells were predicted using an unbiased ligand-receptor interaction analysis. Proteins secreted by tumour cells were analysed by performing mass spectrometry on conditioned medium from patient derived organoid cultures. The conditioned medium was concentrated using a 3kDa Millipore filter and prepared for LC-MS. Mass spectrometry data were acquired in data-dependent acquisition mode. For determining suppression, healthy donor PBMCs were stimulated with anti-CD28/anti-CD3 Dynabeads in the presence of recombinant proteins or concentrated conditioned medium from neuroblastoma organoids. In vitro killing assays were performed with GFP and luciferase transduced organoids and healthy donor PBMCs. Results Analysis of scRNA-seq of 25 neuroblastoma tumours showed a negative correlation between MIF and MDK expression and the cytotoxicity of NK cells, CD8 and gd-T cells within the tumour. These findings could be confirmed with a previously published SEQC bulk-RNAseq dataset containing 498 patients. Next to that, a higher expression of MIF and MDK correlated with poor survival in the same dataset. In the secretome from cultured neuroblastoma organoids, we have used mass spectrometry to identify MIF and MDK amongst the top 100 most abundant proteins from a total of ~1200 proteins. In vitro, we showed that rMIF and rMDK have a suppressive effect on the activation of T cells and the amount of cytotoxic factors such as granzyme B and Perforin are produced by the T cells. This confirms our hypothesis that MIF and MDK negatively influence the cytotoxicity of T cells. Conclusions Using two different unbiased analyses -scRNA-seq analysis of tumours and mass spectrometry of neuroblastoma organoid secretome-, we have identified MIF and MDK as immunosuppressive factor in neuroblastoma. Currently, we are testing several MIF and MDK inhibitors to test if T cell mediated killing of neuroblastoma can be increased if the immunosuppressive MIF and MDK are blocked.