Dendritic Cell-Triggered Immune Activation Goes along with Provision of (Leukemia-Specific) Integrin Beta 7-Expressing Immune Cells and Improved Antileukemic Processes

Abstract

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.

Volatile Phases Derived from Serum, DC, or MLC Culture Supernatants to Deduce a VOC-Based Diagnostic Profiling Strategy for Leukemic Diseases

“…To evaluate and quantify phenotypes of DC/DC leu , leukemic blasts, monocytes and immune reactive cell subsets of the adaptive and innate immunity analyses were conducted via flow cytometry, using a fluorescence-activating cell-sorting flow cytometer (FACSCalibur TM ). Using a refined gating technique and the analysis software CellQuestPro (Becton Dickinson, Heidelberg, Germany), the functionalities of cells (proliferation, cytokine production, degranulation and cytotoxicity) could be investigated [ 12 , 15 ]. Panels with various monoclonal antibodies (moAbs) labelled with fluorescein isothiocyanate (FITC), phycoerythrin (PE), phycoerythrin cyanin 7 (PCy7) or allophycocyanin (APC) were used, provided by Becton Dickinson, Heidelberg, Germany a , Beckman Coulter, Krefeld, Germany b , Santa Cruz Biotechnology, Heidelberg, Germany c , and Bio Legend, San Diego, CA, USA d .…”

“…Antigen-specific response and generation of immunologic memory are the central tasks of the adaptive immune system [ 12 , 13 ]. After activation, naive T cells (T naive , CD3+CD45RO−) are probably converted to non-naive T cells (T non-naive , CD3+CD45RO+), which mediate various immune responses or develop into long-living central memory cells (T cm , CD3+CD45RO+CD197+) or effector memory cells (T em/eff , CD3+CD45RO+CD197−) to facilitate a faster reactivation of the immune system against recurring antigens [ 8 , 15 ]. These immunoreactive cells can be detected using flow cytometry (abbreviations are given in Table 1 ).…”

“…The degranulation assay (Deg) allows the detection and quantification of lysosomal-associated membrane glycoproteins (LAMPs), such as LAMP-1 (CD107a), which are involved in granzyme/perforin-associated degranulation granules. The intracellular cytokine assay (InCyt) allows the intracellular (antigen specific) quantification of cytokines (interferon gamma (INFy) and tumor necrosis factor alpha (TNFa) on a single-cell level, representing triggers of immune responses and mediators of cell apoptosis [ 12 , 15 , 21 ]. Cytotoxicity assays (CTX) evaluate the antileukemic potential of stimulated and unstimulated effector cells [ 12 ].…”

“…Analyses of different (activating or inhibitory) cellular/humoral, soluble factors, or even the smallest molecules could contribute to our understanding of leukemia-related as well as antileukemic processes [ 8 , 15 , 21 , 22 , 23 , 24 ]. In recent years, the role of physical factors (e.g., physiological hypoxia [ 25 ] or circulating vesicles (e.g., extracellular vesicles (EVs) [ 26 , 27 ]) has been tested with respect to a refined monitoring of immunological or tumor-associated processes.…”

Dendritic Cell and Cancer Therapy

“…In particular, the priming method can convert a hard tumor microenvironment to a hot tumor microenvironment and increase DC vaccine efficacy. Lastly, Rackl et al [ 8 ] investigated whether stimulating immune cells in DCs of acute myeloid leukemia (AML) patients and healthy donors leads to increased leukemia-specific integrin beta-7 (ITGβ7) expression in immune cells. ITGβ7 is a subunit of the integrin receptor expressed on the surface of immune cells, mediating cell–cell adhesions, and is involved in an anti-tumor immune response.…”