The Quality and Quantity of Leukemia-derived Dendritic Cells From Patients With Acute Myeloid Leukemia and Myelodysplastic Syndrome Are a Predictive Factor for the Lytic Potential of Dendritic Cells-primed Leukemia-Specific T Cells

Abstract

Adoptive immunotherapy is an important therapy option to reduce relapse rates after stem-cell transplantation in patients suffering from acute myeloid leukemia and myelodysplastic syndromes. Myeloid leukemic cells can regularly be induced to differentiate into leukemia-derived dendritic cells (DC(leu)), regaining the stimulatory capacity of professional dendritic cells (DCs) while presenting the known/unknown leukemic antigen repertoire. So far, induced antileukemic T-cell responses are variable or even mediate opposite effects. To further elicit DC/DC(leu)-induced T-cell-response patterns, we generated DC from 17 Acute myeloid leukemia (AML) and 2 myelodysplastic syndrome cases and carried out flowcytometry and (functional) nonradioactive fluorolysis assays before/after mixed lymphocyte cultures of matched (allogeneic) donor T cells (n=6), T cells prepared at relapse after stem-cell transplantation (n=4) or (autologous) patients' T cells (n=7) with blast containing mononuclear cells ("MNC") or DC(leu) ("DC"). Compared with "MNC", "DC" were better mediators of antileukemic-activity, although not in every case effective. We could define DC subtypes and cut-off proportions of DC subtypes/qualities (mature DC/DC(leu)) after "DC" priming, which were predictive for an antileukemic activity of primed T cells and the clinical course of the disease after immunotherapy (allogeneic stem-cell transplantation/donor lymphocytes infusion/therapy). In summary, our data show that the composition and quality of DC after a mixed lymphocyte culture-priming phase is predictive for a successful ex vivo antileukemic response, especially with respect to proportions of mature and leukemia-derived DC. These data contribute not only to predict DC-mediated functions or the clinical course of the diseases but also to develop and refine DC-vaccination strategies that may pave the way to develop and modify adoptive immunotherapy, especially for patients at relapse after allogeneic stem-cell transplantation.

Cytokine Release Patterns in Mixed Lymphocyte Culture (MLC) of T-Cells with Dendritic Cells (DC) Generated from AML Blasts Contribute to Predict anti-Leukaemic T-Cell Reactions and Patients’ Response to Immunotherapy

“…For an improvement of therapy of AML-/MDS-pts at relapse after SCT GM-CSF might be applied in the context of DLI in order to facilitate the generation of APC from leukaemic blasts and thereby to increase antileukaemic activities of donor T-cells against myeloid blasts, as already shown by others and us in ex vivo settings (Kufner et al 2005;Kremser et al 2010;Schmid et al 2011;Dreyßig et al 2011). We could already show that compared to 'MNC'-stimulation stimulation of T-cells with 'DC'/DC leu , as effective APCs presenting the complete leukaemic antigenic repertoire, gives rise to a better mediation of an antileukaemic T-cell response in vitro thereby potentially contributing to a better response to DLI after allogeneic SCT in vivo (Kufner et al 2005;Kremser et al 2010;Grabrucker et al 2010;Dreyßig et al 2011;Buhmann et al 2016). Those 'DC'-/DC leu -stimulated T-cells could be promising candidates for adoptive immunotherapies in selected pts (Schmetzer & Schmid 2012).…”

“…Recently our group has shown, that T-cell stimulation with 'DC' is more efficient than 'MNC'-stimulation with respect to a gain of antileukaemic activity of those T-cells Grabrucker et al 2010). Our data show, that compared to cytokine levels in AML-pts' serum increased levels of all cytokines were found in MLC-culture supernatants of T-cells (independent whether autologous, allogeneic or harvested after SCT) stimulated either with 'MNC' or 'DC', pointing to induced T-cell activation and growth (Houssiau & Van Snick 1992;Kammula et al 1999).…”

“…Correlation of anti-leukaemic lytic T-cell activities with cytokine release patterns after stimulation of T-cells with 'MNC' and 'DC' (Figure 2) We evaluated the role of soluble factors at second restimulation of T-cells stimulated with 'DC' or 'MNC' and at cell harvest of MLC Grabrucker et al 2010;Schmetzer & Schmid 2012). Tcell sources were either 'allogeneic', 'autologous' or from pts at relapse 'after SCT'.…”

“…Proportions of positive events in defined gates compared with the isotype controls were calculated using Cell Quest software (Becton Dickinson, BD, Heidelberg, Germany). Quantification and characterization of DC and DC leu were performed by Flow cytometry according to our gating strategy already described by former groups (Schmetzer et al 2007;Kremser et al 2010;Liepert et al 2010;Grabrucker et al 2010;Dreyßig et al 2011). Flow cytometric analyses were carried out on day 0 and 10 followed by a functional cytotoxic fluorolysis assay on day 10 as described below.…”

“…Half medium exchange was performed on day 3 and 6 of culture. Cells were harvested after 10 days of co-culture and the cytotoxicity fluorolysis assay was carried out as described Grabrucker et al 2010). For analysis and quantification of lymphocytes, monocytes and leukaemic blasts before culture the total MNC fraction was gated.…”