Influence of physiological hypoxia on whole blood (WB) from healthy donors and AML-patients: the (leukemic) antigen-presentation on blasts, on leukemia-derived dendritic cells (DCleu) after stimulation with "DC-generating cocktails" as well as the antileukemic functions of DC/ DCleu-stimulated immune reactive cells

Doraneh Gard, Fatemeh (2021): Influence of physiological hypoxia on whole blood (WB) from healthy donors and AML-patients: the (leukemic) antigen-presentation on blasts, on leukemia-derived dendritic cells (DCleu) after stimulation with "DC-generating cocktails" as well as the antileukemic functions of DC/ DCleu-stimulated immune reactive cells. Dissertation, LMU München: Medizinische Fakultät

Abstract

DCleu generation in vivo and transferring to the patients could qualify as a novel treatment option. Furthermore, blasts modulated to leukemia-derived DC (DCleu) in vivo could result in T-cell activation. We have already established a method using a combination of GM-CSF+ PICIBANIL (I), or +PGE2 (K) or + PGE1 (M) kits to convert leukemic blasts into DCleu without induction of blasts’ proliferation in a WB-culture model (in the presence of individual patients’ soluble and cellular components). Such DCleu cells can strongly induce antileukemic and memory T-cells. To simulate physiological conditions, we compared Normoxic (No) standard- (21% O2) vs. physiological Hypoxic (Hy) (10% O2) culture conditions. In the first part of my work, DC-generation was done in AML cell lines to set up the physiological conditions. AML cell lines were cultured with immunomodulatory Kits (-I, -K, -M) under Normoxic condition (21% Oxygen) as well as under Hy (approx. 6% Oxygen). Our cell line results showed no significant changes in DCleu (frequencies of DCleu/Bla+) or the proliferation of blasts in Hy condition under the stimulation of Kits compared to control and also as compared to No condition. In the second part of my work, samples from AML-patients and healthy blood donors (peripheral blood mononuclear cells (PBMNC) and heparinized whole blood (WB)) were cultured in parallel under No (21% Oxygen) and Hy (10% Oxygen) using Pici-PGE1, Pici-PGE2 and Kit-I, Kit-K and Kit-M DC-generating media which followed by MLC (mixed lymphocyte culture) using patients’ or healthy donors’ T-cells and functional (blast-cytotoxicity- and cytokine secretion) assays in AML-samples. Compositions of DC-, blast-subtypes, the immune status, and results of functional assays were compared for all tested conditions. We found that DCs and their subtypes can be generated in comparable frequencies from AML and healthy PBMNCs samples using pici-PGE1 and pici-PGE2 under No and Hy conditions. Comparable frequencies of DC were generated with Kits (-I, -K, -M) from 35 AML and 16 healthy WB -samples. DC generation was comparable under No vs. Hy in AML and healthy samples. Further, as control when there was no added response modifier (w/o), no or low production of DC/DCleu was detected. Interestingly, no induction of blasts’ proliferation was shown under the influence of kits under No or Hy conditions. After MLC of T-cells with kit-treated AML-WB under No vs. Hy (with Kit-I, -K, -M), we found a significant increase of activated T-cells, CD4 +CD3+/CD3+, Tprol, Tnon-naive, Teff-em as well as of NK and iNKT cells and low frequencies of Treg, Tcm DC, T-cells, NK, iNKT, and CIK cells after MLC under Hy as well as after No conditions. Finally, we demonstrated an increase of antileukemic activity after the stimulation of patients’ T-cells in MLC with (DC/DCleu containing) Kit-I or -M treated (vs. untreated) WB in No as well as under Hy conditions. Average blast lysis was increased in MLCWB-DC Kit-I and MLCWB-DC Kit-M compared to MLCWB, with equal average lysis in MLCWB-DC Kit-I and MLCWB-DC Kit-M. We further found significantly higher amounts of INFγ secreting cells of the innate and adaptive immune system after MLCWB-DC Kit-I and MLCWB-DC Kit-M compared to MLCWB. Results were comparable under No and Hy conditions. We found a significant positive correlation between the DC/WB produced with Kit-M and the antileukemic reactivity of T ‘effector-cells’ after MLCWB-DC kit-M. Furthermore, we found a significant correlation between DC+/WB and DCleu /WB generated with kit-M and the best anti-leukemic reactivity of T-cells’ MLCWB-DC kit-M in Hy conditions.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: AML, Acute myeloid leukemia, Dendritic cells, Hypoxic condition, Patient, Whole blood
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 16. Dezember 2021
• 1. Bericht­erstatter:in: Schmetzer, Helga