Natural killer cells and immune control: Deciphering the transcription factors that regulate expression of the NKG2D-ligand MICA

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NKG2D (encoded by the KLRK1 gene, killer cell lectin-like receptor subfamily K, member 1) is a C-type lectin receptor and an important activating immunoreceptor involved in tumor surveillance. NKG2D is expressed on the surface of most cytotoxic lymphocytes such as NK cells, CD8+ T cells, some γδ T cells, and possibly also some CD4+ T cells and known as a sensor for damaged or dangerous cells. Ligands for NKG2D (NKG2D-L) are generally not expressed on healthy cells but are induced on the surface of malignant cells. Evasion from the innate NKG2D-mediated immune surveillance is a hallmark of many solid and hematopoietic tumors and depends among other strategies on proteolytic shedding and release of soluble NKG2D-L. This renders target cells invisible to an NKG2D-dependent NK cell attack and moreover causes a downregulation of receptor surface expression on effector cells. Targeting the NKG2D-NKG2D-L axis to stimulate anti-tumor immunity is a promising therapeutic approach. Here, we aim to identify specific transcription factors, which regulate the inducible expression of MHC Class I Polypeptide-Related Sequence A (NKG2D-L, MICA). Until today the network of transcription factors regulating the up-regulation of MICA is not defined. A better understanding of factors that direct NKG2D-L expression will contribute to the development of novel therapeutic strategies aiming at an increased NKG2D-L expression on the tumor cell surface and thus more efficient killing by NK cells. In our previous work, we demonstrated that CBP/p300 acetyltransferases and the cAMP responsive element binding protein (CREB) contribute critically to the inducible expression of MICA. Using an enChIP approach we identified several transcription factors which are in response to histone deacetylase inhibition associated with the active MICA promoter including Krüppel-like factor 4 (KLF4), Yin Yang 1 (YY1), and CCCTC-Binding Factor (CTCF). The aim of this project is to assess the involvement of these candidate proteins, in particular transcription factors, in the regulation of MICA expression. For this purpose, we plan to analyze to what extend these factors regulate the transcription of MICA in promoter-reporter assays. Further overexpression and depletion approaches are planned to investigate the impact on MICA surface protein expression and on NK cell-mediated target cell elimination. Finally the clinical relevance of the identified factors will be assessed in acute myeloma leukemia patient samples. To this end we will perform Rhapsodytm (Beckman Coulter) single-cell sequencing to correlate MICA and candidate factor expression and to measure whether these factors regulate (gain/loss of function) MICA expression and susceptibility against NK cells. We expect that we will identify positive regulators of MICA which may on the long run represent novel therapeutic targets.