P01.10 IFNy secretion of adaptive and innate immune cells as a parameter to display leukaemia derived dendritic cell (DC_leu) mediated immune responses in AML

Abstract

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.

Influence of probiotics on the periodontium, the oral microbiota and the immune response during orthodontic treatment in adolescent and adult patients (ProMB Trial): study protocol for a prospective, double-blind, controlled, randomized clinical trial

“…Analyses of frequencies of innate and adaptive immune cells: Peripheral blood mononuclear cells (PBMNC) will be separated from whole blood samples by density gradient centrifugation using the Ficoll-Hypaque technique (Biocoll separating solution; Biochrom, Berlin, Germany), washed, suspended in phosphate-buffered saline (PBS; Biochrom, Berlin, Germany) and frozen according to standardized protocols [ 81 , 83 – 85 ]. The cellular immune status will be analysed quantitatively and qualitatively (frequencies of immune cells and their subtypes in the PBMNC-fraction) with thawed PBMNC using multiparametric flow cytometry (Cytoflex S, Beckman Coulter, Indianapolis, United States) [ 81 , 83 – 85 ].…”

“…Analyses of frequencies of innate and adaptive immune cells: Peripheral blood mononuclear cells (PBMNC) will be separated from whole blood samples by density gradient centrifugation using the Ficoll-Hypaque technique (Biocoll separating solution; Biochrom, Berlin, Germany), washed, suspended in phosphate-buffered saline (PBS; Biochrom, Berlin, Germany) and frozen according to standardized protocols [ 81 , 83 – 85 ]. The cellular immune status will be analysed quantitatively and qualitatively (frequencies of immune cells and their subtypes in the PBMNC-fraction) with thawed PBMNC using multiparametric flow cytometry (Cytoflex S, Beckman Coulter, Indianapolis, United States) [ 81 , 83 – 85 ]. Lymphocyte typing will be performed using an ‘inflammatory panel’ to quantify different innate and adaptive immune cells, e.g., different CD4 or CD8 T cell subtypes (activated, proliferating/non-naïve/memory (T pro , T non-naïve, T mem ), T helper cells (Th 1 /Th 2 /Th 17 cells) or regulatory T cells (T regs ), B cells, natural killer (NK) cells, natural killer T (iNKT) cells and dendritic cells (DC) [ 81 , 83 , 84 ].…”

“…The cellular immune status will be analysed quantitatively and qualitatively (frequencies of immune cells and their subtypes in the PBMNC-fraction) with thawed PBMNC using multiparametric flow cytometry (Cytoflex S, Beckman Coulter, Indianapolis, United States) [ 81 , 83 – 85 ]. Lymphocyte typing will be performed using an ‘inflammatory panel’ to quantify different innate and adaptive immune cells, e.g., different CD4 or CD8 T cell subtypes (activated, proliferating/non-naïve/memory (T pro , T non-naïve, T mem ), T helper cells (Th 1 /Th 2 /Th 17 cells) or regulatory T cells (T regs ), B cells, natural killer (NK) cells, natural killer T (iNKT) cells and dendritic cells (DC) [ 81 , 83 , 84 ].…”