Strategies to optimize natural killer cell functions in the tumor microenvironment

Immune cell-based therapies are currently changing the oncological treatment landscape worldwide. While genetically modified T cells have already been approved for clinical use in few hematological cancers, natural killer (NK) cell-based therapies are employed in early phase clinical trials. NK cells are generally regarded as an alternative to T cells as they possess the advantage of intrinsic killing capacity of malignant cells, cause less side effects post transplantation and can be used in an allogeneic setting. In the context of multiple myeloma (MM), the second most common blood cancer, two chimeric antigen receptor (CAR) T cell products are clinically approved and NK cell-based therapies show promising results in early phase clinical studies. However, relapses occur after CAR-T cell therapy and although NK cells show a good safety profile, it is evident that genetic modifications and combination therapies are needed to improve their efficacy. The research provided in this thesis aims to elaborate strategies to improve NK cell functionality in the tumor microenvironment (TME) through genetic modification and combination with an oncolytic virus, with a special focus on MM. It is well established that immune cell dysfunctionality can occur through chronic stimulation via the programmed death protein 1 (PD1) pathway. Blocking the interaction of PD1 with its ligands PD-L1 and PD-L2 restores immune cell effector functions and is successfully applied in several hematological and solid malignancies. However, in MM, immune checkpoint inhibition has failed due to lack of efficacy in monotherapies and severe side effects in combination therapies. Therefore, paper I explores the feasibility of novel NK cell specific chimeric switch receptors (CSR) that re-direct inhibitory signaling into activating signaling in NK cells upon binding of PD1 ligands. The designed CSR are based on the extracellular domain of PD1 that is linked to the transmembrane and/or intracellular domains of DAP10, DAP12 and CD3z which are known to provide NK cell activation. In paper I, we show that primary PD1-CSR+ NK cells exert superior cytotoxicity and cytokine release against PD-L1+ cancer cell lines and primary autologous MM samples, establishing them as a promising strategy towards PD-L1+ cancers. Targeting CD38 with the monoclonal antibody (mAb) daratumumab has improved treatment outcomes for patients with both newly diagnosed and relapsed or refractory MM, but is also met with resistance to therapy. Moreover, the majority of peripheral blood NK cells express CD38 and are depleted during daratumumab treatment. In paper II, we have introduced an affinity-optimized CD38-CAR construct into CD38dim primary NK cells that naturally occur during a long-term cytokine-based feeder cell-free expansion protocol. This approach leads to a functional NK cell product with superior cytotoxicity against CD38+ MM cell lines and autologous MM samples with minimal risk for fratricide. The data provided here pave the way for advancing this approach as an alternative treatment option for MM. Oncolytic viruses (OV) present an attractive immunotherapeutic platform for combination therapies and are currently tested in pre-clinical and early phase clinical trials for several malignancies. Herpes simplex virus 1 (HSV-1) based OV belong to the most utilized viruses and were shown to effectively infect and lyse malignant MM cells. However, their clinical applicability is met with several challenges, one being a rapid clearance by the immune system. NK cells naturally recognize virally infected cells and have been implicated in both promoting and hampering OV therapy. However, the exact molecular mechanism remain elusive. In paper III we have studied the interaction of primary NK cells with target cells that have been infected with the HSV-1 based OV HSV1716. The data provided in paper III show that primary NK cells increase their degranulation ability against HSV1716-infected target cells, despite the downregulation of important ligands for activating NK cell receptors. Primary NK cells become infected with HSV1716 through direct contact with target cells and increase their activation status. A better understanding of the molecular mechanisms behind NK cell recognition of HSV-1 OV infected target cells might improve immunotherapeutic combination approaches. All in all, the constituting studies provide unique and novel data on how to improve NK cell-based immunotherapies for cancer. Although the main focus is laid on MM, the presented approaches can be expanded to other malignancies in order to improve NK cell therapies and increase NK cell fitness in an immunosuppressive TME

Chromosome identification and reconstruction of evolutionary rearrangements in Brachypodium distachyon, B. stacei and B. hybridum

Background and Aims The Brachypodium genus represents a useful model system to study grass genome organization. Palaeogenomic analyses (e.g. Murat F, Armero A, Pont C, Klopp C, Salse J. 2017. Reconstructing the genome of the most recent common ancestor of flowering plants. Nature Genetics 49: 490–496) have identified polyploidization and dysploidy as the prime mechanisms driving the diversity of plant karyotypes and nested chromosome fusions (NCFs) crucial for shaping grass chromosomes. This study compares the karyotype structure and evolution in B. distachyon (genome Bd), B. stacei (genome Bs) and in their putative allotetraploid B. hybridum (genomes BdBs). • Methods Brachypodium chromosomes were measured and identified using multicolour fluorescence in situ hybridization (mcFISH). For higher resolution, comparative chromosome barcoding was developed using sets of low-repeat, physically mapped B. distachyon-derived bacterial artificial chromosome (BAC) clones. • Key Results All species had rather small chromosomes, and essentially all in the Bs genome were morphometrically indistinguishable. Seven BACs combined with two rDNA-based probes provided unambiguous and reproducible chromosome discrimination. Comparative chromosome barcoding revealed NCFs that contributed to the reduction in the x = 12 chromosome number that has been suggested for the intermediate ancestral grass karyotype. Chromosome Bd3 derives from two NCFs of three ancestral chromosomes (Os2, Os8, Os10). Chromosome Bs6 shows an ancient Os8/Os10 NCF, whilst Bs4 represents Os2 only. Chromosome Bd4 originated from a descending dysploidy that involves two NCFs of Os12, Os9 and Os11. The specific distribution of BACs along Bs9 and Bs5, in both B. stacei and B. hybridum, suggests a Bs genome-specific Robertsonian rearrangement. • Conclusions mcFISH-based karyotyping identifies all chromosomes in Brachypodium annuals. Comparative chromosome barcoding reveals rearrangements responsible for the diverse organization of Bd and Bs genomes and provides new data regarding karyotype evolution since the split of the two diploids. The fact that no chromosome rearrangements were observed in B. hybridum compared with the karyotypes of its phylogenetic ancestors suggests prolonged genome stasis after the formation of the allotetraploid

Impact of Chromosomal Rearrangements on the Interpretation of Lupin Karyotype Evolution

Plant genome evolution can be very complex and challenging to describe, even within a genus. Mechanisms that underlie genome variation are complex and can include whole-genome duplications, gene duplication and/or loss, and, importantly, multiple chromosomal rearrangements. Lupins (Lupinus) diverged from other legumes approximately 60 mya. In contrast to New World lupins, Old World lupins show high variability not only for chromosome numbers (2n = 32–52), but also for the basic chromosome number (x = 5–9, 13) and genome size. The evolutionary basis that underlies the karyotype evolution in lupins remains unknown, as it has so far been impossible to identify individual chromosomes. To shed light on chromosome changes and evolution, we used comparative chromosome mapping among 11 Old World lupins, with Lupinus angustifolius as the reference species. We applied set of L. angustifolius-derived bacterial artificial chromosome clones for fluorescence in situ hybridization. We demonstrate that chromosome variations in the species analyzed might have arisen from multiple changes in chromosome structure and number. We hypothesize about lupin karyotype evolution through polyploidy and subsequent aneuploidy. Additionally, we have established a cytogenomic map of L. angustifolius along with chromosome markers that can be used for related species to further improve comparative studies of crops and wild lupins

Sensitivity of microwave radiometers with square - law and linear detectors, Journal of Telecommunications and Information Technology, 2004, nr 1

Stochastic analysis of modulation microwave radiometers with square - law and linear detectors is presented in the paper. Assuming ideal detector characteristics it is shown that in typical applications, i.e., in very low power measurements, a type of detector used is of no influence on total radiometer sensitivity. Other aspects of use of a particular detector are also presented

Multifrequency microwave thermograph for biomedical applications, Journal of Telecommunications and Information Technology, 2004, nr 1

This paper presents problems related to thermal radiation of human bodies in microwave range in aspect of diagnosis of breast carcinoma. A mathematical model of thermal radiation transfer through tissues is introduced and methods of measurement of temperature, depth and size of a heat source, by means of multifrequency microwave thermography are described. Theoretical considerations are supplemented by presentation of experimental results

Genetic and Methylome Variation in Turkish Brachypodium Distachyon Accessions Differentiate Two Geographically Distinct Subpopulations

Brachypodium distachyon (Brachypodium) is a non-domesticated model grass species that can be used to test if variation in genetic sequence or methylation are linked to environmental differences. To assess this, we collected seeds from 12 sites within five climatically distinct regions of Turkey. Seeds from each region were grown under standardized growth conditions in the UK to preserve methylated sequence variation. At six weeks following germination, leaves were sampled and assessed for genomic and DNA methylation variation. In a follow-up experiment, phenomic approaches were used to describe plant growth and drought responses. Genome sequencing and population structure analysis suggested three ancestral clusters across the Mediterranean, two of which were geographically separated in Turkey into coastal and central subpopulations. Phenotypic analyses showed that the coastal subpopulation tended to exhibit relatively delayed flowering and the central, increased drought tolerance as indicated by reduced yellowing. Genome-wide methylation analyses in GpC, CHG and CHH contexts also showed variation which aligned with the separation into coastal and central subpopulations. The climate niche modelling of both subpopulations showed a significant influence from the “Precipitation in the Driest Quarter” on the central subpopulation and “Temperature of the Coldest Month” on the coastal subpopulation. Our work demonstrates genetic diversity and variation in DNA methylation in Turkish accessions of Brachypodium that may be associated with climate variables and the molecular basis of which will feature in ongoing analyses. View Full-Text Keywords: Brachypodium; DNA methylation; drought; flowering; genome; phenomic

Small but crucial : the novel small heat shock protein Hsp21 mediates stress adaptation and virulence in Candida albicans

Peer reviewedPublisher PD