<b><i>Introduction:</i></b> Myeloid leukaemic blasts can be converted into leukaemia-derived dendritic cells (DC_leu), characterised by the simultaneous expression of dendritic- and leukaemia-associated antigens, which have the competence to prime and enhance (leukaemia-specific) immune responses with the whole leukaemic antigen repertoire. To display and further specify dendritic cell (DC)- and DC_leu-mediated immune responses, we analysed the interferon gamma (IFNy) secretion of innate and adaptive immune cells. <b><i>Methods:</i></b> DC/DC_leu were generated from leukaemic whole blood (WB) with (blast)modulatory Kit-I (granulocyte-macrophage colony-stimulating factor [GM-CSF] + Picibanil [OK-432]) and Kit-M (GM-CSF + prostaglandin E1) and were used to stimulate T cell-enriched immunoreactive cells. Initiated anti-leukaemic cytotoxicity was investigated with a cytotoxicity fluorolysis assay. Initiated IFNy secretion of T, NK, CIK, and iNKT cells was investigated with a cytokine secretion assay (CSA). IFNy positivity was additionally evaluated with an intracellular cytokine assay (ICA). Recent activation of leukaemia-specific cells was verified through addition of leukaemia-associated antigens (LAA; WT-1 and Prame) <b><i>Results:</i></b> We found Kit-I and Kit-M competent to generate mature DC and DC_leu from leukaemic WB without induction of blast proliferation. Stimulation of immunoreactive cells with DC/DC_leu regularly resulted in an increased anti-leukaemic cytotoxicity and increased IFNy secretion of T, NK, and CIK cells, pointing to the significant role of DC/DC_leu in leukaemia-specific alongside anti-leukaemic reactions. Interestingly, an addition of LAA did not further increase IFNy secretion, suggesting an efficient activation of leukaemia-specific cells. Here, both the CSA and ICA yielded comparable frequencies of IFNy-positive cells. Remarkably, the anti-leukaemic cytotoxicity positively correlated with the IFNy secretion in T^CD3+, T^CD4+, T^CD8+, and NK^CD56+ cells. <b><i>Conclusion:</i></b> Ultimately, the IFNy secretion of innate and adaptive immune cells appeared to be a suitable parameter to assess and monitor the efficacy of in vitro and potentially in vivo acute myeloid leukaemia immunotherapy. The CSA in this regard proved to be a convenient and reproducible technique to detect and phenotypically characterise IFNy-secreting cells. In respect to our studies on DC-based immunomodulation, we were able to display the potential of DC/DC_leu to induce or improve leukaemia-specific and anti-leukaemic activity.

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BackgroundMyeloid leukaemic blasts can be converted into leukaemia derived dendritic cells (DCleu) with blastmodulatory Kit-I and Kit-M, which have the competence to regularly activate T and immunoreactive cells to gain anti-leukaemic activity or rather cytotoxicity. As innate and adaptive immune responses are notably promoted by the cytokine interferon gamma (IFNy), we hypothesised that the IFNy secretion could be a suitable parameter to display DC/DCleu mediated immunologic activity and even anti-leukaemic cytotoxicity.Materials and MethodsDC/DCleu were generated from leukaemic WB with Kit-I (GM-CSF + OK-432) and Kit-M (GM-CSF + PGE1) and used to stimulate T cell enriched immunoreactive cells. Initiated anti-leukaemic cytotoxicity was investigated with a cytotoxicity fluorolysis assay (CTX). Initiated IFNy secretion of innate and adaptive immune cells (T cells, TCD4+ cells, TCD8+ cells, NKCD56+ cells, NKCD161+ cells, CIKCD56+ cells, CIKCD161+ cells and iNKT) was investigated with a cytokine secretion assay (CSA). In some cases IFNy production was additionally evaluated with an intracellular cytokine assay (ICA). Conclusively, the IFNy secretion of immunoreactive cells was correlated with the anti-leukaemic cytotoxicity.ResultsSignificant amounts of DC and DCleu as well as migratory DC and DCleu could be generated with Kit-I and Kit-M without induction of blast proliferation. T cell enriched immunoreactive cells stimulated with DC/DCleu showed an increased anti-leukaemic cytotoxicity and an increased IFNy secretion of T, NK and CIK cells compared to control. Both the CSA and ICA yielded comparable amounts of IFNy positive innate and adaptive immune cells. The correlation between the IFNy secretion of immunoreactive cells and the anti-leukaemic cytotoxicity showed a positive relationship in T cells, TCD4+ cells, TCD8+ cells and NKCD56+ cells.ConclusionsWe found blastmodulatory Kit-I and Kit-M competent to generate DC/DCleu from leukaemic WB. Stimulation of T cell enriched immunoreactive cells with DC/DCleu regularly resulted in an increased anti-leukaemic cytotoxicity and an increased IFNy dependent immunological activity of T, NK and CIK cells compared to control. Moreover the anti-leukaemic cytotoxicity positively correlated with the IFNy secretion in T cells, TCD4+ cells, TCD8+ cells, NKCD56+ cells. We therefore consider the IFNy secretion of innate and adaptive immune cells to be a suitable parameter to assess the efficacy of in vitro and potentially in vivo AML immunotherapy. The CSA in this regard proved to be a convenient and reproducible technique to detect and phenotypically characterise IFNy secreting cells of the innate and adaptive immune system.Disclosure InformationL.K. Klauer: None. O. Schutti: None. S. Ugur: None. F. Doraneh-Gard: None. N. Rogers: None. M. Weinmann: None. D. Krämer: None. A. Rank: None. C. Schmid: None. B. Eiz-Vesper: None. H.M. Schmetzer: None.