Mechanisms of activation and cytotoxicity in γδ T cells and natural killer cells
Authors: Kuhnigk Kyra 
Published: February 2025

<p dir="ltr">γδ T cells and natural killer (NK) cells have been shown to play crucial roles in the immune response. While the activation mechanisms of these two cell types differ, with γδ T cells being activated by small metabolites from the isoprenoid synthesis pathway and NK cells through balancing signals between activating and inhibitory receptors, their effector functions were believed to resemble each other. Both cell types are well known for their quick release of IFN-γ and TNF-α and for their cytotoxic responses to infected and malignant cells. Consequently, both γδ T cells and NK cells are regarded as promising candidates for immunotherapies. However, the outcomes of clinical trials conducted thus far remain inconsistent, suggesting the presence of a lack of fundamental knowledge. To enhance our understanding of these multifaceted immune cells, this thesis aimed to address several key research questions. We explored the cytotoxic pathways employed by γδ T cells and NK cells, trying to identify the factors that limit their effectiveness. We further examined how activation affects their potency and employed advanced imaging techniques to investigate NK cell infiltration in tumour spheroids. To achieve this, we employed a range of single-cell microscopy-based approaches to elucidate the heterogeneity of γδ T cell and NK cell populations.</p><p dir="ltr">We found that following a three-day period of stimulation, γδ T cells exhibited an upregulation of activating receptors, with a particularly pronounced increase in granzyme B expression. However, after the proliferation and expansion of the γδ T cells, there was a decline in granzyme B expression levels. Investigation of the cytotoxic mechanisms employed by γδ T cells revealed that they predominantly utilise degranulation as their primary mode of action, with the engagement of death receptors playing a comparatively minor role. Further, early activated γδ T cells with high levels of granzyme B used degranulation more often than the late expanded γδ T cells. However, the substantial quantities of granzyme B did not facilitate the serial killing of multiple target cells, as γδ T cells only expressed low levels of perforin.</p><p dir="ltr">In addition, we established that γδ T cells were efficiently activated by monocytes through the sensing of phosphoantigens as well as cytokines. These cells responded by releasing IFN-γ and TNF-α within hours of these interactions and formed close contacts with monocytes for extended periods. The investigation of these close contacts revealed that calcium responses in γδ T cells reached a peak at approximately 10 hours, which coincided with the maturation and potential accumulation of IPP in monocytes.</p><p dir="ltr">NK cells are known to be effective killers, capable of eliminating numerous target cells in succession. Here we investigated the factors that disable NK cells from continuing to execute their function. We revealed that even after killing of several target cells NK cells retained a reserve of lytic granules. Instead of experiencing a depletion of their lytic cargo, these cells appeared to be receiving an insufficient level of signalling. However, upon stimulation with novel stimuli, even those NK cells that had ceased to kill were able to resume their function.</p><p dir="ltr">Finally, to enhance our understanding of infiltrating NK cells, an expansion microscopy approach was utilised to enhance imaging of NK cells in tumour spheroids. This technique has the potential to be employed in future studies to combine functional live cell assays with high-resolution imaging of infiltrating immune cell.</p><p dir="ltr">In summary, this thesis contributes to a better understanding of the fundamental biology in γδ T cells and NK cells, particularly their cytotoxic functions. These findings are of critical importance in the development of more effective immune therapies.</p><h3>List of scientific papers</h3><p dir="ltr">I. P. A. Sandoz*, <b>K.</b> <b>Kuhnigk*</b>, E. K. Szabo, S. Thunberg, E. Erikson, N. Sandström, Q. Verron, A. Brech, C. Watzl, A. K. Wagner, E. Alici, K.J. Malmberg, M. Uhlin and B. Önfelt. Modulation of lytic molecules restrain serial killing in γδ T lymphocytes. Nature Communications. 14, 6035 (2023). <a href="https://doi.org/10.1038/s41467-023-41634-7" rel="noreferrer" target="_blank">https://doi.org/10.1038/s41467-023-41634-7</a></p><p dir="ltr">II. <b>K. Kuhnigk</b>, H. Zhang, A. Stikvoort, P. A. Sandoz* and B. Önfelt *. Activation dynamics of γδ T cells in response to monocyte interaction. [Manuscript]</p><p dir="ltr">III. H. van Ooijen, Q. Verron, <b>K. Kuhnigk</b>, P. A. Sandoz, N. Sandström and B. Önfelt. Loss of signaling limits NK cell serial killing. [Submitted]</p><p dir="ltr">IV. S. J. Edwards, V. Carannante, <b>K. Kuhnigk</b>, H. Ring, T. Tararuk, F. Hallbook, H. Blom, B. Önfelt and H. Brismar. High-Resolution Imaging of Tumor Spheroids and Organoids Enabled by Expansion Microscopy. Frontiers in Molecular Biosciences. 7:208 (2020). <a href="https://doi.org/10.3389/fmolb.2020.00208" rel="noreferrer" target="_blank">https://doi.org/10.3389/fmolb.2020.00208</a></p><p dir="ltr">*Authors contributed equally</p>