In vitro Untersuchung spenderspezifischer Unterschiede in der antileukämischen Lysefähigkeit von NK-Zellen gegenüber pädiatrischer B-Zell-Leukämien mit und ohne CD19 Antikörper

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Dokumentart: PhDThesis
Date: 2020-10-26
Language: German
Faculty: 4 Medizinische Fakultät
Department: Medizin
Advisor: Lang, Peter (Prof. Dr.)
Day of Oral Examination: 2020-09-25
DDC Classifikation: 610 - Medicine and health
Keywords: Leukämie , Immunologie , Antikörper
Other Keywords: NK-Zellen
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The treatment of refractory and relapsed B-lineage precursor leukemia (ALL) has improved by advancements in allogeneic SCT and CD19-targeting approaches. KIR/HLA interaction has been shown absolutely relevant for the survival of patients suffering from leukemia. This was shown in AML and childhood ALL in haploidentical SCT. Fc modified antibodies can enhance ADCC efficiently irrespective of KIR geno- and phenotype and FCγIIIa polymorphism. We analyzed the influence of activatory and inhibitory receptors on NK-Cells using an Fc-optimized monoclonal CD19 antibody. NK profiling of healthy donors by KIR genotyping, KIR phenotyping and assessment of NKG2D receptor, DNAM-1 coreceptor, NCRs and PD-1 receptor was done by CD107a assay and cytotoxicity assays using primary PBMCs and preactivated NK with and without the CD19 mAb 4G7SDIE. Different BCP-ALL cell lines (n=4, NALM-16, NALM-6, MHH-cALL-4 and SEM) were immune characterized for HLA-I-genotype and quantitative HLA-I expression, NKG2D ligand, DNAM-1 ligand and CD19-expression as to be the most prominent parameters to determine NK cell mediated recognition, cytolysis and ADCC in CD19+ BCP-ALL. Additionally, the influence of PD-1 receptor-ligand interaction was assessed. Further experiments included upregulation and blockade of HLA-I, blockade of NKG2D-R and DNAM-1-R. All donors showed low antileukemic activity of PBMCs. Preactivation and expansion of donors' PBMCs significantly increased activation of NK cells and cytolysis. In all patients coincubation of CD19 mAb led to enhanced cytolysis and NK cell degranulation as well as IFNγ production. RL-mismatch and NK education showed an impact on NK activity. Irrespective of the BCP-ALL cell line, superior and inferior donors were identified by in vitro testing partly controversial to KIR genotyping results. In conclusion, we demonstrate that defined tumor-specific features determine NK cell recognition, cytolysis and ADCC. Further, donor-specific characteristics indentify donors for their NK Cells activity, cytolysis and ADCC implicating to extend donor selection strategies by functional effector cell assessment. The role of checkpoint inhibitors in NK alloreactivity of BCP-ALL needs further evaluation.

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