Expression von immunreaktiven NKG2DL in der gesunden und malignen Hämatopoese

Natural killer group 2, member D ligands (NKG2DL) are known immunogenic molecules, whose cell surface presentation is absent in healthy cells but is induced following cellular stress or malignant transformation, thereby rendering cells susceptible to immune surveillance by NKG2D receptor (NKG2DR) expressing NK and cytotoxic T cells. Absence of NKG2DL on the cell surface enables immune evasion and is reported in various cancers including acute myeloid leukemia (AML), where our laboratory could previously show that reduced NKG2DL expression is a feature of leukemic stem cells (LSC). Here I aim to investigate the intracellular mechanisms inducing NKG2DL surface presentation in both malignant and healthy cells.
Using RT-qPCR, western blotting, ELISA, image, and conventional flow cytometry, I analyzed NKG2DL expression on mRNA and protein levels and determined the intracellular localization, surface presentation and shedding of NKG2DL in AML cells, healthy hematopoietic cells from cord blood (CB) or adult peripheral blood, as well as genetically modified CB HSPC expressing mixed lineage leukemia (MLL) fusion proteins.
I show for the first time that NKG2DL are expressed intracellularly with or without subsequent released into the extracellular fluids in malignant and healthy hematopoietic cells. Interestingly, similar levels of NKG2DL mRNA and protein were detected in healthy HSPC, MLL rearranged (MLLr) CB HSPC, and AML cell lines, indicating a mechanism controlling the intracellular retention of NKG2DL that could importantly contribute to the regulation of NKG2DL surface expression.
Surprisingly, when compared to MLL1 gene breakpoints at intron 9, breakpoint localization at intron 11 did not, or only partially allow NKG2DL surface presentation. Absence of NKG2DL in MLL-AF4 (Intron 11) subpopulations coincided with an increased clonogenic potential similar to previous reports in primary AML. Additionally, I show that current AML and potential future therapies (PARP1 and GATA2 inhibition) do not affect NKG2DL surface presentation in healthy cells while treatment with IFN γ increases NKG2DL cell surface expression of MLL-AF4 (Intron 11) cord blood derived cells.
Here, I present an approach to systematically characterize NKG2DL presentation in both healthy and malignant hematopoietic cells, showing for the first time that NKG2DL are robustly expressed in healthy hematopoietic cells, but retained intracellularly. Mechanisms regulating intracellular NKG2DL retention may enable rapid induction of NKG2DL surface expression and thereby immediate immune clearance of damaged cells. Crucially, an improved understanding of NKG2DL presentation and retention in these malignant cells could lead to novel strategies for targeting therapy resistant cancer cells such as AML LSC.