Natural killer cell development and function in the context of allogenic stem cell transplantation and cell therapy

Abstract

Natural killer (NK) cells play a crucial role in the control of viral infections and transformed, cancerous cells. These innate cells have high cytotoxic capacities and use a multitude of receptors to interact with neighbouring cells. The balance of activating and inhibitory signals derived from these receptors determines if NK cells release this cytotoxic potential towards the encountered cell or not. It is this high receptor diversity that defines the phenotype and function of discrete NK cell subsets. This thesis focused on investigating some of the multiple dimensions that influence the function and development of NK cells, with the ultimate aim of improving the current approaches to NK cell immunotherapy against haematological malignancies.

NK cell education, the process by which NK cells are effectively trained to recognise the body’s own HLA molecules on healthy cells, is instrumental to generate highly cytotoxic NK cells. When educated NK cells encounter cells missing these self HLA-ligands they lose their inhibition, and the signalling balance is shifted towards activation and killing of the encountered cell. This process is greatly influenced by the interaction of inhibitory KIR receptors and particular HLA molecules. Paper I explores the intimate relationship between allelic variation and NK cell function. It delves into how the polymorphism in KIR and HLA-class I genes influences the loading of granzyme B, a key effector molecule harboured in the secretory lysosomes of NK cells. Employing a high-resolution analysis of these genes' variations across a population of 365 blood donors, we observed that allelic variation in HLA-class I genes is responsible for the observed variation in granzyme B levels, which, in turn, plays a critical role in determining the lytic hit and downstream killing of MHC-deficient target cells. We further showed that NK cell education is stable over time and is independent of age or gender. These findings underscore the significant role genetic factors play in influencing NK cell functionality, laying the groundwork for subsequent research and potential therapeutic developments that could leverage these genetic influences.

Haematopoietic stem cell transplantation (HSCT) is a potentially curative treatment for multiple haematological malignancies. A quick and effective reconstitution of the NK cell compartment following transplantation has been shown to correlate with better clinical outcomes and lower relapse incidence. However, immunosuppressive compounds need to be given to the patient to keep the grafted immune system in check and avoid GvHD. Sirolimus, an mTOR inhibitor, has been recognized as an effective component of GvHD prophylaxis regimens. Paper II analyses the effect of sirolimus containing GvHD prophylaxis on the immune reconstitution of transplantation patients. Employing a broad immune cell mapping technique, we uncovered a profound suppression of the naïve CD4 T cell compartment while relatively sparing regulatory T cells and enriching activated CD8 T cells. Given NK cells’ important antileukemic potential, we focused our evaluation on NK cell phenotype, function, and proliferative capacity. The results show that treatment with sirolimus leads to similar immune cell profiles as conventional prophylaxis but leaves a significant impact on NK cell reconstitution. The sirolimus-treated group showed a slight maturation of NK cell subsets and a reduction in their proliferation, and differentiation. This work provides insights into the lasting effects of mTOR inhibition on immune reconstitution and specifically on NK cells following HSCT. And highlight the importance of metabolic regulation in therapeutically modulating NK cell responses during HSCT.

The field of NK cell immunotherapy has expanded dramatically in the last decades, however identifying suitable sources of highly potent NK cells has remained a challenge. Allogeneic NK cell therapy, exploiting the missing-self effect of donor educated NK cells to boost their killing potential towards the patients’ leukemic cells, has been shown to have a high potential of inducing remission. Adaptive NK cells have been shown to be a highly educated and potent NK cell subset. In Paper III we developed a GMP-compliant protocol for expanding adaptive NK cells to generate large doses of highly functional and alloreactive ADAPT-NK cells. We demonstrate that ADAPT-NK retain similar transcriptomic and phenotypic features as conventional adaptive NK cells and are potent killers of a wide array of HLA-C/KIR mismatched tumour cell lines and primary AML samples. Finally, combination with NK cell engagers to re-direct their killing broadens their applicability beyond HLA-C/KIR mismatch. The results of this study define a path towards an effective NK cell-based therapy for haematological malignancies and introduces the concept of an ‘’off-the-shelf’’ cell therapy with a non-engineered yet highly specific NK cell population.

Collectively, this thesis provides a multi-dimensional exploration of NK cell development and function. It underscores the significant role of genetic variation in modulating NK cell functionality, reveals the lasting impact of mTOR inhibition on NK cell reconstitution following HSCT, and highlights the therapeutic potential of using specially expanded adaptive NK cells for treating haematological malignancies. These insights expand our understanding of NK cell biology in transplantation and cell therapy and could contribute to developing novel therapeutic strategies for treating haematological diseases.